SIRT1和 SIRT6之间的协同作用有助于识别 DNA 断裂,并加强人类和小鼠的 DNA 损伤反应和修复

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Synergy between SIRT1 and SIRT6 helps recognize DNA breaks and potentiates the DNA damage response and repair in humans and mice

Abstract

摘要

The DNA damage response (DDR) is a highly orchestrated process but how double-strand DNA breaks (DSBs) are initially recognized is unclear. Here, we show that polymerized SIRT6 deacetylase recognizes DSBs and potentiates the DDR in human and mouse cells. First, SIRT1 deacetylates SIRT6 at residue K33, which is important for SIRT6 polymerization and mobilization toward DSBs. Then, K33-deacetylated SIRT6 anchors to γH2AX, allowing its retention on and subsequent remodeling of local chromatin. We show that a K33R mutation that mimics hypoacetylated SIRT6 can rescue defective DNA repair as a result of SIRT1deficiency in cultured cells. These data highlight the synergistic action between SIRTs in the spatiotemporal regulation of the DDR and DNA repair in humans and mice.

DNA 损伤反应(DDR)是一个高度协调的过程,但双链 DNA 断裂(dsb)是如何最初识别的尚不清楚。在这里,我们表明,高分子化的 SIRT6去乙酰化酶识别 dsb 并在人和小鼠细胞中增强 DDR。首先,SIRT1在残基 K33上对 SIRT6进行脱乙酰化,这对 SIRT6的聚合和向 dsb 的动员起重要作用。然后,k33去乙酰化的 SIRT6锚定到 γh2ax 上,使其保留在局部染色质上并随后重塑。我们发现一个 K33R 基因突变模拟低乙酰化的 SIRT6可以挽救培养细胞 SIRT1缺陷引起的 DNA 修复缺陷。这些数据突出表明 sirt 在 DDR 的时空调节和 DNA 修复在人类和小鼠中的协同作用。“Open annotations. The current annotation count on this page is打开注释。此页面上的当前注释计数为0.Introduction 引言

DNA damage can be induced by various endogenous and exogenous agents. Upon detection of damage, the DNA damage response (DDR) is immediately elicited to regain genomic integrity via chromatin remodeling, signaling transduction and amplification (Ciccia and Elledge, 2010). Double-strand breaks (DSBs) are the most severe type of DNA lesion; they are recognized by the Mre11-Rad50-Nbs1 (MRN) complex, which recruits and activates phosphatidylinositol 3-kinase-like protein kinase ataxia-telangiectasia mutated (ATM) or ATM- and Rad3-related (ATR). H2AX is then rapidly phosphorylated (γH2AX) by ATM/ATR, and serves as a platform to localize repair proteins near to the DNA breaks (Celeste et al., 2003). Simultaneously, various histone-modifying enzymes, heterochromatin factors and ATP-dependent chromatin remodelers work cooperatively to relax the chromatin structure and ensure that additional repair factors have access to the DSBs (Price and D’Andrea, 2013). Despite all these advances in understanding the DDR, how DSBs are initially and precisely recognized is largely unknown.

DNA 损伤可以由多种内源性和外源性因素引起。一旦检测到损伤,DNA 损伤反应(DDR)立即启动,通过染色质重塑、信号转导和扩增恢复基因组完整性(Ciccia and Elledge,2010)。双链断裂(dsb)是最严重的 DNA 损伤类型,它们被 Mre11-Rad50-Nbs1(MRN)复合体所识别,该复合体能够招募和激活磷脂酰肌醇3激酶样蛋白激酶共济失调-毛细血管扩张变异(ATM)或 ATM-和 rad3相关(ATR)。然后,H2AX 被 ATM/ATR 快速磷酸化(γH2AX) ,并作为一个平台定位修复 DNA 断裂附近的蛋白质(Celeste 等人,2003)。同时,各种组蛋白修饰酶、异染色质因子和 atp 依赖性染色质重塑协同工作,放松染色质结构,确保其他修复因子能够接触到 DSBs (Price 和 d’ andrea,2013)。尽管在理解 DDR 方面取得了这些进步,但是 dsb 最初是如何被精确地识别的,这在很大程度上还是未知的。

NAD+-dependent sirtuins belong to class III histone deacetylases (HDACs) (Houtkooper et al., 2012). Seven sirtuins (SIRT1-7) with various enzymatic activities and physiological functions are expressed in mammals. SIRT1, 6 and 7 localize in the nucleus and seem to be most relevant to the DDR as their depletion causes growth retardation, a defective DDR and DNA repair and premature aging (Mostoslavsky et al., 2006Wang et al., 2008Vazquez et al., 2016). Upon DNA damage, SIRT1 redistributes on chromatin, co-localizes with γH2AX, and deacetylates XPA, NBS1 and Ku70 to regulate nucleotide excision repair, homologous recombination (HR) and non-homologous end-joining (NHEJ) (Fang et al., 2016Yuan et al., 2007Fan and Luo, 2010Jeong et al., 2007). Depleting Sirt1 in mouse fibroblasts impairs the DDR and leads to genomic instability (Wang et al., 2008). SIRT6 is one of the earliest factors recruited to DSBs; it initiates the subsequent recruitment of SNF2H, H2AX, DNA-PKcs and PARP1 (Atsumi et al., 2015McCord et al., 2009Van Meter et al., 2016). SIRT6 mono-ribosylates PARP1 to enhance its activity (Mao et al., 2011). Despite their rapid mobilization to DNA breaks, the triggers for sirtuin recruitment are obscure (Vazquez et al., 2016Dobbin et al., 2013Toiber et al., 2013).

依赖 NAD + 的去乙酰化酶属于 III 类组蛋白去乙酰化酶(HDACs)。哺乳动物中表达了7种具有多种酶活性和生理功能的去乙酰化酶(SIRT1-7)。SIRT1、6和7位于细胞核内,并且似乎与 DDR 最相关,因为它们的损耗会导致生长迟缓、 DDR 和 DNA 修复缺陷以及过早衰老(Mostoslavsky 等人,2006; Wang 等人,2008; Vazquez 等人,2016)。DNA 损伤后,SIRT1在染色质上重新分布,与 γh2ax 共定位,并去乙酰化 XPA、 NBS1和 Ku70以调节核苷酸切除修复、同源重组(HR)和非同源末端连接(NHEJ)(Fang et al. 2016; Yuan et al. 2007; Fan and Luo,2010; Jeong et al. 2007)。耗尽小鼠成纤维细胞中的 Sirt1损害 DDR 并导致基因组不稳定(Wang 等人,2008)。SIRT6是 dsb 招募的最早因素之一; 它启动了随后 SNF2H、 H2AX、 DNA-PKcs 和 PARP1的招募(Atsumi 等人,2015; McCord 等人,2009; Van Meter 等人,2016)。SIRT6单核糖基化 PARP1以增强其活性(Mao 等人,2011)。尽管它们迅速动员到 DNA 断裂,但是去乙酰化酶补充的触发机制是模糊的(Vazquez 等人,2016; Dobbin 等人,2013; Toiber 等人,2013)。

Here, we aimed to delineate the mechanisms underlying DSB recognition. We found that SIRT6 polymerizes and directly recognizes DSBs via a putative DNA-binding pocket consisting of N- and C-termini from two adjacent molecules. SIRT1 interacts with SIRT6 and deacetylates it at K33, thus allowing its polymerization and recognition of DSBs. A K33R mutant, mimicking hypoacetylated SIRT6, could rescue DNA repair defects in SIRT1 knockout (KO) cells. Our data highlight an essential synergy between SIRT1 and SIRT6 in the spatiotemporal regulation of the DDR.

在这里,我们的目的是描述机制的基础 DSB 识别。我们发现 SIRT6通过一个由两个相邻分子的 n-和 c- 末端组成的假定的 dna 结合口袋聚合并直接识别 DSBs。SIRT1与 SIRT6相互作用,并在 K33脱乙酰,因此允许其聚合和识别双链。模拟低乙酰化 SIRT6的 K33R 突变体能够修复 SIRT1基因敲除(KO)细胞的 DNA 修复缺陷。我们的数据强调了 SIRT1和 SIRT6在 DDR 的时空调节中的重要协同作用。Results 结果

SIRT6 directly recognizes DNA double-strand breaks

SIRT6能直接识别 DNA 双链断裂

Nuclear SIRTs (SIRT1/6/7) are quickly mobilized to DSBs (Figure 1—figure supplement 1) and serve as a scaffold for DNA repair factors (Vazquez et al., 2016Dobbin et al., 2013Toiber et al., 2013). Intriguingly, these nuclear SIRTs are also activated by RNA and the nucleosome (Gil et al., 2013Tong et al., 2017). We thus reasoned that SIRTs might directly sense DNA breaks, especially DSBs. To test our hypothesis, we established a molecular docking simulation using AutoDock Vina software (Trott and Olson, 2010). We obtained the crystal structures for SIRT1 (PDB code 4I5I) (Zhao et al., 2013), SIRT6 (PDB code 3PKI) (Pan et al., 2011) and SIRT7 (PDB code 5IQZ) (Priyanka et al., 2016) from the Protein Data Bank (https://www.rcsb.org). We removed the heteroatoms to expose interaction regions and added Gasteiger charges to construct docking models. A DSB structure was extracted from the PDB code 4DQY (Langelier et al., 2012). As SIRTs use NAD+ as a co-substrate in amide bond hydrolysis, which shares a similar skeleton to the phosphate, base and ribose groups on broken DSB ends, we included NAD+ as a simulation control.

核 SIRTs (SIRT1/6/7)被迅速移植到 dsb 中(图1ー图补充1) ,并作为 DNA 修复因子的支架(Vazquez et al. ,2016; Dobbin et al. ,2013; Toiber et al. ,2013)。有趣的是,这些核 sirt 也被 RNA 和核小体激活(Gil 等人,2013; Tong 等人,2017)。因此,我们推断 sirt 可能直接感知 DNA 断裂,特别是 dsb。为了验证我们的假设,我们使用 AutoDock Vina 软件(Trott 和 Olson,2010)建立了分子对接模拟。我们从蛋白质数据库中获得了 SIRT1(PDB 代码4I5I)(Zhao et al. ,2013) ,SIRT6(PDB 代码3PKI)(Pan et al. ,2011)和 SIRT7(PDB 代码5IQZ)(Priyanka et al. ,2016)的晶体结构。我们去掉了杂原子,暴露了相互作用区域,并添加了 Gasteiger 电荷来构建对接模型。DSB 结构是从 PDB 代码4DQY (Langelier et al. ,2012)中提取的。利用 NAD + 作为酰胺键水解的共基底,在 DSB 断裂端上与磷酸基团、碱基团和核糖基团具有相似的骨架结构,采用 NAD + 作为模拟对照。

We found that the binding affinity between NAD+ and all nuclear SIRTs was within the range of –eight to –10 kcal/mol (Figure 1A). Surprisingly, only the binding between the DSB and SIRT6 was energetically favored (–12.7 kcal/mol), being even lower than that of NAD+ (Figure 1A,B). This finding suggested that SIRT6 might directly bind to DSBs and prompted us to gain further experimental evidence.

我们发现 NAD + 与所有核 SIRTs 之间的结合亲和力在 -8-10 kcal/mol 范围内(图1A)。令人惊讶的是,只有 DSB 和 SIRT6之间的结合是积极的(- 12.7 kcal/mol) ,甚至低于 NAD + (图1 a,b)。这一发现表明,SIRT6可能直接与 dsb 结合,并促使我们获得进一步的实验证据。Figure 1 图1 with 1 supplement 还有一种补充剂Download asset下载资产Open asset开放资产

SIRT6 directly recognizes DNA breaks.SIRT6可以直接识别 DNA 碎片(A) The predicted binding affinity (kcal/mol) between sirtuins (SIRTs) and ligands (NAD+ and a DSB) by molecular docking analysis. (B) Molecular docking of SIRT6 with a DSB (right) and NAD+ (left) … see more(a)分子对接分析预测了 sirtuins (SIRTs)与配体(NAD + 和 DSB)之间的结合亲和力(kcal/mol)。(b) SIRT6与 DSB (右)和 NAD + (左)的分子对接… 详见

Figure 1—figure supplement 1 图1ー图补充资料1Download asset下载资产Open asset开放资产

DSB-recruitment kinetics of SIRTs.特定结构吸引子的招募动力学(A) GFP-fused SIRT1, SIRT6 and SIRT7 were introduced into mouse embryonic fibroblasts (MEFs). The fluorescence signal was captured at 20 s and 1 min after laser-induced DNA damage. Scale bar, 10 μm. … see more(a)将 gfp 融合的 SIRT1、 SIRT6和 SIRT7导入小鼠胚胎成纤维细胞(MEFs)。荧光信号在激光诱导 DNA 损伤后20s 和1min 被捕获。比例尺,10微米。看到更多

We next generated a DSB-mimicking biotin-conjugated DNA duplex and performed an in vitro pulldown assay. Recombinant SIRT6 (rSIRT6), but not rSIRT1 or rSIRT7, bound to the DNA duplex (Figure 1C). This finding was verified by a fluorescence polarization (FP) assay using a FAM-labeled DNA duplex. We observed dynamic FP (Kd = 166.3 nM; Figure 1D), supporting a specific and direct interaction between the DNA duplex and rSIRT6. By contrast, the FP was minimal for rSIRT1, rSIRT7 and GST control (Figure 1D). To interrogate whether such binding is specific to broken DNA, we repeated the pulldown assay in the presence of unlabeled linear or circular DNA. While linearized DNA inhibited rSIRT6 binding to the DNA duplex, circular DNA had a minimal effect (Figure 1E). Together, these data indicate that SIRT6, but not SIRT1 or SIRT7 recognizes and directly binds to DSBs.

我们接下来产生了一个模拟 dsb 的生物素结合 DNA 双链,并在体外进行了下拉实验。重组 SIRT6(rSIRT6) ,但不是 rSIRT1或 rSIRT7,结合到 DNA 双链(图1C)。这一发现是由荧光偏振(FP)分析使用一个 fam-labled DNA 双链证实。我们观察到动态 FP (Kd = 166.3 nM; 图1D) ,支持 DNA 双链和 rSIRT6之间的特异性和直接的相互作用。相比之下,rSIRT1、 rSIRT7和 GST 控制的 FP 最小(图1D)。为了确定这种结合是否是断裂 DNA 特有的,我们在未标记的线性或圆形 DNA 存在的情况下重复了下拉试验。线性化的 DNA 抑制 rSIRT6与 DNA 双链的结合,环状 DNA 的作用微乎其微(图1E)。总之,这些数据表明 SIRT6,而不是 SIRT1或 SIRT7识别并直接绑定到 dsb。

SIRT6 is dynamically acetylated in the N terminus at K33

SIRT6在 K33的 n 端动态乙酰化

As predicted from the crystallographic data, SIRT6s form an asymmetric hexamer (Pan et al., 2011) that generates three potential DSB binding pockets; each of these pockets consist of two N-termini and two C-termini from two adjacent molecules (Figure 2—figure supplement 1A). Both the N-termini and C-termini are essential for SIRT6 to associate with chromatin (Tennen et al., 2010). To gain biochemical evidence for SIRT6 polymerization, we employed a biomolecule fluorescence compensation system (BiFC). In brief, we cloned SIRT6 cDNA into either the N-terminal or C-terminal of a yellow fluorescence protein (YFP), herein termed N-SIRT6 and C-SIRT6. The yellow fluorescence was detectable by FACS only when N-SIRT6 directly interacted with C-SIRT6. After co-transfecting these constructs into HEK293 cells, we detected a strong fluorescence signal by FACS in >24% cells (Figure 2—figure supplement 1B), suggesting a direct interaction between the two SIRT6 molecules. By contrast, fluorescence signal was rarely detected in BiFC analysis of N-SIRT6 and C-SIRT3 or non-tagged SIRT6 and C-SIRT3 (Figure 2—figure supplement 1C). To confirm this SIRT6 polymerization event, we performed co-immunoprecipitation (Co-IP) in HEK293 cells in which we had co-overexpressed FLAG-SIRT6 and HA-SIRT6. Consistently, we detected FLAG-SIRT6 but not FLAG-SIRT3 in the anti-HA-SIRT6 immunoprecipitates (Figure 2—figure supplement 1D).

根据晶体学数据预测,SIRT6s 形成了一个非对称的六聚体(Pan 等人,2011) ,产生了三个潜在的 DSB 结合口袋,每个口袋由两个相邻分子的 N-termini 和两个 C-termini 组成(图2ー图补充1A)。N 末端蛋白和 c 末端蛋白都是 SIRT6与染色质结合所必需的(Tennen 等人,2010)。为了获得 SIRT6聚合反应的生物化学依据,我们采用了生物分子荧光补偿系统(BiFC)。简单地说,我们将 SIRT6的 cDNA 克隆到一个黄色荧光蛋白(YFP)的 n 端或 c 端,这里称为 n-SIRT6和 c-SIRT6。只有当 N-SIRT6与 C-SIRT6直接相互作用时,流式细胞仪才能检测到黄色荧光。共转染 HEK293细胞后,流式细胞仪(FACS)检测到大于24% 的细胞(图2ー图1B)出现强荧光信号,提示两个 SIRT6分子之间存在直接相互作用。与此相反,在 N-SIRT6和 C-SIRT3的 BiFC 分析中,或者在未标记的 SIRT6和 C-SIRT3(图2ー图1C)中,荧光信号很少被检测到。为了证实这种 SIRT6聚合事件,我们在共表达了 flag-SIRT6和 ha-SIRT6的 HEK293细胞中进行了共免疫沉淀(Co-IP)。在抗 ha-sirt6的免疫沉淀物中,我们一直检测到 FLAG-SIRT6,但没有检测到 FLAG-SIRT3(图2ー图1D)。

The DSB phosphate backbone is negatively charged. The positive-charged environment of SIRT6 thus favors its binding to DSBs. Indeed, one of our predicted DSB-binding pockets formed by two adjacent molecules in SIRT6 hexamer consisted of six positively charged residues at the edge, namely four arginine (R32/39) and two lysine (K33) residues (Figure 2—figure supplement 1E). Acetylation is the most redundant post-translational modification that converts positively charged K to neutral Kac. This property is utilized by proteins with a lysine-rich domain (KRD), for example Histones, Ku70 and p53, for mediating dynamic interactions with proteins harboring an acidic domain like SET (Wang et al., 2016). The heterodimerized Ku70 and Ku80 complex directly senses DSBs via a flexible C-termini containing multiple K residues, and regulates NHEJ (Hu et al., 2012). We therefore examined whether SIRT6 is (de)acetylated on these K residues thus sensing DSBs. We immunoprecipitated FLAG-SIRT6 with an anti-FLAG antibody and then probed the immunoprecipitate with anti-Kac antibodies. As shown, Kac was detected in the precipitated FLAG-SIRT6 immunocomplex (Figure 2A). We then purified FLAG-SIRT6 and performed high-resolution LC-MS/MS to identify which K residues are acetylated (Supplementary file 1). We identified K15 and K33 in the N-terminus. To confirm these acetylated K residues, we generated K15R and K33R point mutants, with K17R serving as a negative control. While neither K15R nor K17R affected the FLAG-SIRT6 acetylation level, K33R significantly inhibited it (Figures 2A and Figure 2—figure supplement 2), supporting that K33 is dynamically (de)acetylated.

DSB 磷酸骨架带负电荷。因此,SIRT6的正电荷环境有利于它与 dsb 的绑定。实际上,我们预测的 SIRT6六聚体中两个相邻分子形成的 dsb 结合口袋中,有一个由边缘六个带正电荷的残基组成,即四个精氨酸(R32/39)和两个赖氨酸(K33)残基(图2ー图1E)。乙酰化是将正电荷 k 转化为中性 Kac 的最冗余的翻译后修饰。这一特性被富含赖氨酸结构域(KRD)的蛋白质所利用,例如组蛋白、 Ku70和 p53,用于介导与包含酸性结构域的蛋白质的动态相互作用,如 SET (Wang 等人,2016)。异质化的 Ku70和 Ku80复合体通过一个包含多个 k 残基的柔性 C-termini 直接感知 DSBs,并调节 NHEJ (Hu 等人,2012)。因此,我们检测了 SIRT6是否在这些 k 残基上(去乙酰化) ,从而感知 dsb。我们用抗 flag 抗体免疫沉淀 FLAG-SIRT6,然后用抗 kac 抗体检测其免疫沉淀物。如图所示,在沉淀的 FLAG-SIRT6免疫复合物中检测到 Kac (图2A)。我们然后纯化了 FLAG-SIRT6,并进行了高分辨液相色谱-串联质谱(LC-MS/MS)鉴定哪些 k 残基被乙酰化(补充文件1)。我们在 n 端鉴定了 K15和 K33。为了证实这些乙酰化 k 残基,我们以 K17R 为阴性对照,生成了 K15R 和 K33R 点突变体。K15R 和 K17R 均不影响 FLAG-SIRT6的乙酰化水平,K33R 对其有明显的抑制作用(图2A 和图2ー图2补充2) ,支持 K33动态去乙酰化。Figure 2 图2 with 4 supplements 有4种补充剂Download asset下载资产Open asset开放资产

SIRT6 K33 (de)acetylation regulates DSB binding.SIRT6 K33(de)乙酰化调节 DSB 结合(A) The acetylation levels of FLAG-SIRT6 WT and K33R were assessed by western blotting with pan-acetyl antibodies in anti-FLAG immunoprecipitates in HEK293T cells. (B) Streptavidin pulldown assay … see more以 HEK293T 细胞中抗 flag 免疫沉淀物的泛乙酰抗体评价 FLAG-SIRT6 WT 和 K33R 的乙酰化水平。(b)链霉亲和素下拉试验… 更多信息

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Polymerization of SIRT6.SIRT6的聚合(A) Yellow fluorescence detection by FACS in HEK293 cells ectopically expressing vector only, SIRT6-WT, SIRT6-KR, SIRT6-KQ or SIRT6-HY via the BiFC system. Green ovals indicate … see more(a)流式细胞仪检测 HEK293细胞外表面表达载体 SIRT6-WT、 SIRT6-KR、 SIRT6-KQ 或 SIRT6-HY 的黄色荧光。绿色椭圆表示… 更多信息

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Deacetylase activity of SIRT6.SIRT6的去乙酰化酶活性(A) Western blotting analysis of the acetylation levels of histone H3 in SIRT6KO HEK293 cells reconstituted with SIRT6 WT or indicated mutants. (B) Cell fractionation analysis to detect the … see more对 SIRT6重组的 KO HEK293细胞组蛋白 h 3乙酰化水平的西方墨点法分析。(b)细胞分馏分析检测… 更多

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Acetylation of SIRT6.SIRT6的乙酰化SIRT6 K15/17R acetylation levels were determined by IP and western blotting with a pan anti-acetyl lysine antibody in HEK293 cells.在 HEK293细胞中,用激光等离子体和西方墨点法分别测定 SIRT6 K15/17R 乙酰化水平,并用泛抗乙酰赖氨酸抗体测定。

Figure 2—figure supplement 1 图2ー图补充1Download asset下载资产Open asset开放资产

Polymerization of SIRT6.SIRT6的聚合(A) Schematic of one putative DSB-binding pocket, consisting of two N-termini (yellow and aquamarine) and two C-termini (orange and lime green) of two adjacent SIRT6 hexamer molecules. (B) Schematic … see more(a)一个假定的 dsb 结合口袋示意图,由两个相邻的 SIRT6六聚体分子的 n 端(黄色和海蓝宝石)和两个 c 端(橙色和石灰绿色)组成。(b)示意图… 更多

Dynamic SIRT6 K33 (de)acetylation regulates DSB sensing

动态 SIRT6 K33(de)乙酰化调节 DSB 传感

To understand the function of SIRT6 K33 acetylation, we examined whether it is required for binding to DSBs. We used our K33R mutant and generated a new K33Q mutant to mimic deacetylated or acetylated SIRT6, respectively (Tang et al., 2017). We also mutated SIRT6 H133 to a tyrosine residue (H133Y) to blunt SIRT6 enzymatic activity (Toiber et al., 2013). K33Q and H133Y binding to the DNA duplex was significantly compromised compared to WT and K33R binding (Figure 2B). Consistently, we recorded notable FP for SIRT6 K33R (Kd = 104.9 nM) but little FP for SIRT6 K33Q (Figure 2C).

为了了解 SIRT6 K33的乙酰化作用,我们研究了 SIRT6 K33与 dsb 的结合是否需要乙酰化。我们使用我们的 K33R 突变体,并产生一个新的 K33Q 突变体,分别模拟去乙酰化或乙酰化的 SIRT6(Tang 等人,2017)。我们还将 SIRT6 H133突变为酪氨酸残基(H133Y) ,以减弱 SIRT6的酶活性(Toiber 等人,2013)。与 WT 和 K33R 结合相比,K33Q 和 H133Y 结合 DNA 双链的能力明显下降(图2B)。同时,SIRT6 K33R (Kd = 104.9 nM)的 FP 值较高,而 SIRT6 K33Q 的 FP 值较低(图2C)。

We then monitored GFP-SIRT6 mobility in cells upon receipt of DNA damage. H133 is critical for enriching SIRT6 on chromatin (Tennen et al., 2010). We reconstituted GPF-SIRT6 WT, K33Q, K33R and H133Y in Sirt6–/– cells and monitored their recruitment to DSBs. While the K33Q and H133Y mutations significantly jeopardized efficient SIRT6 recruitment to DNA breaks, the SIRT6 K33R mutant retained such ability (Figure 2D,E). To gain more experimental support, we made use of an inducible DR-GFP reporter system that contains a unique I-SceI cutting site. In presence of triamcinolone acetonide (TA), the I-SceI-GR enzyme translocated to the nucleus within 10 min and generated DSBs, as evidenced by an increase in the γH2AX level (Figure 2F,G). We thus monitored the occupancy of SIRT6 on chromatin surrounding these induced DSBs, by chromatin immunoprecipitation (ChIP) and quantitative PCR, as previously described (Soutoglou et al., 2007). Both the K33Q and H133Y mutations compromised SIRT6 recruitment to the sites of damage, whereas SIRT6 K33R recruitment was comparable to that of SIRT6 WT (Figure 2H).

然后,我们监测 GFP-SIRT6的流动性,在细胞接受 DNA 损伤。H133是丰富染色质 SIRT6的关键(Tennen et al. 2010)。我们在 Sirt6-/-细胞中重组 GPF-SIRT6 WT、 K33Q、 K33R 和 H133Y,并监测它们在 dsb 中的招募。虽然 K33Q 和 H133Y 突变显著损害了有效的 SIRT6补充 DNA 断裂,但 SIRT6 K33R 突变保留了这种能力(图2D,e)。为了获得更多的实验支持,我们使用了一个可诱导的 DR-GFP 报告系统,其中包含一个独特的 I-SceI 切割位点。在有曲安奈德(TA)存在的情况下,I-SceI-GR 酶在10分钟内转移到细胞核并产生 DSBs,γh2ax 水平的增加证明了这一点(图2F,g)。因此,我们监测了 SIRT6在这些诱导的 dsb 周围的染色质占有率,使用染色质免疫沉淀和定量 PCR,正如前面描述的(Soutoglou 等人,2007)。K33Q 和 H133Y 突变都损害了 SIRT6对损伤部位的补充,而 SIRT6 K33R 补充与 SIRT6 WT 的补充相当(图2H)。

Upon DNA damage, the acetylation levels of H3K9 and H3K56 decline, and after repair, goes back to the original level (Tjeertes et al., 2009). H3K9ac and H3K56ac are deacetylating targets of SIRT6, indicating that SIRT6 might contribute to the reduced H3K9ac and H3K56ac levels on the DSB-surrounding chromatin. Indeed, reconstituted SIRT6 WT and K33R downregulated the levels of H3K9ac and H3K56ac in Sirt6–/– cells, while K33Q and H133Y failed (Figure 2I and J and Figure 2—figure supplement 3A). Further, the K33Q and H133Y mutations also affected SNF2H recruitment to DSBs (Figure 2—figure supplement 3B), which requires SIRT6 (Toiber et al., 2013), but no effect was observed in the presence of the K33R mutation. Of note, neither K33R nor K33Q affected the deacetylase activity of SIRT6 (Figure 2—figure supplement 3C).

DNA 损伤后,H3K9和 H3K56的乙酰化水平下降,修复后,回到原来的水平(Tjeertes 等人,2009年)。H3K9ac 和 H3K56ac 是 SIRT6的去乙酰化靶点,提示 SIRT6可能有助于 dsb 周围染色质 H3K9ac 和 H3K56ac 水平的降低。实际上,重组的 SIRT6 WT 和 K33R 降低了 SIRT6/-细胞中 H3K9ac 和 H3K56ac 的水平,而 K33Q 和 H133Y 则失效(图2I 和 j 以及图2ー图3A)。此外,K33Q 和 H133Y 突变也影响了 SNF2H 对 dsb 的补充(图2ー图3B) ,这需要 SIRT6(Toiber 等人,2013) ,但没有观察到 K33R 突变的影响。值得注意的是,K33R 和 K33Q 均不影响 SIRT6的去乙酰化酶活性(图2ー图3C)。

We next analyzed whether dynamic K33 (de)acetylation modulates SIRT6 polymerization. We co-overexpressed HA-SIRT6 and various FLAG-SIRT6 mutants and performed Co-IP. We detected FLAG-SIRT6 in the anti-HA immunoprecipitates, supporting that SIRT6 polymerization occurs (Figure 2—figure supplement 1D). While HA-SIRT6 was still able to bind to FLAG-SIRT6 K33R, its binding to SIRT6 K33Q was significantly jeopardized. Of note, the enzyme-dead H133Y mutation also jeopardized SIRT6 polymerization. This finding is consistent with the fact that the H133 site is important for both SIRT6 deacetylase activity and for mediating the chromatin association (Tennen et al., 2010). We confirmed this jeopardized polymerization in the K33Q mutant condition by BiFC assay (Figure 2—figure supplement 4A,B). Together, these data implicate that dynamic SIRT6 K33 (de)acetylation modulates SIRT6 polymerization and thus DSB binding.

接着分析了动态 K33(去)乙酰化是否调节 SIRT6聚合反应。我们共表达 HA-SIRT6和各种 FLAG-SIRT6突变体,并进行了 Co-IP。我们在抗 ha 的免疫沉淀物中检测到了 FLAG-SIRT6,支持 SIRT6发生聚合(图2ー图1D)。虽然 HA-SIRT6仍然能够与 FLAG-SIRT6 K33R 结合,但其与 SIRT6 K33Q 的结合受到严重损害。值得注意的是,酶死 H133Y 突变也危及 SIRT6的聚合。这一发现与 H133位点对于 SIRT6去乙酰化酶活性和介导染色质结合的重要性是一致的(Tennen 等人,2010)。我们通过 BiFC 实验(图2ー图4A,b)证实了在 K33Q 突变体条件下的聚合危害性。综上所述,这些数据表明动态的 SIRT6 K33(去)乙酰化可以调节 SIRT6的聚合,从而与 DSB 结合。

SIRT6 interacts with SIRT1

SIRT6与 SIRT1相互作用

Having confirmed SIRT6 (de)acetylation, we moved to examine potential deacetylase of SIRT6. To this end, we first tested the effect of various HDAC inhibitors on SIRT6 acetylation level. We noticed that the level of acetylated SIRT6 was largely elevated in the presence of the class III HDAC (SIRTs) inhibitor nicotinamide (NAM) or the SIRT1-specific inhibitor Ex527, but not the class I/II HADC inhibitor Trichostatin A (TSA) (Figure 3—figure supplement 1). This finding suggested that SIRT1 might be involved in SIRT6 deacetylation. Indeed, co-IP and western blotting revealed that FLAG-SIRT6 interacted with endogenous SIRT1 (Figure 3A) and vice versa in HEK293 cells (Figure 3B). In addition, we detected SIRT1 in the anti-SIRT6 immunoprecipitates and vice versa(Figure 3C,D). A GST pulldown assay confirmed that His-SIRT1 directly interacted with GST-SIRT6 (Figure 3E). Further, we observed co-localization between SIRT6 and SIRT1 by confocal microscopy in cells co-transfected with GFP-SIRT6 and DsRed-SIRT1 or in cells co-stained with specific antibodies (Figure 3F and Figure 3—figure supplement 2A).

在确认了 SIRT6(去)乙酰化之后,我们进一步研究了 SIRT6的潜在去乙酰化酶。为此,我们首先测试了各种 HDAC 抑制剂对 SIRT6乙酰化水平的影响。我们注意到,在含有 III 类 HDAC 抑制剂(SIRTs)烟酰胺(NAM)或 sirt1特异性抑制剂 Ex527的情况下,乙酰化的 SIRT6水平明显升高,而 I/II 类 HADC 抑制剂曲古抑菌素 a (TSA)(图3ー图补充1)没有升高。提示 SIRT1可能参与了 SIRT6的脱乙酰化反应。实际上,co-IP 和西方墨点法表明,在 HEK293细胞中,FLAG-SIRT6与内源性 SIRT1相互作用,反之亦然。另外,我们在抗 sirt6的免疫沉淀物中检测到 SIRT1,反之亦然(图3C,d)。GST 下拉实验证实 His-SIRT1与 GST-sirt6直接相互作用(图3E)。进一步,我们观察到 SIRT6和 SIRT1在与 gfp-SIRT6和 dsred-SIRT1共转染的细胞中,或在特异性抗体共染的细胞中,通过共聚焦显微镜共定位 SIRT1(图3F 和图3ー图2A)。Figure 3 图3 with 2 supplements 还有两种补充剂Download asset下载资产Open asset开放资产

SIRT6 interacts with SIRT1.SIRT6与 SIRT1相互作用(A) Western blot analysis of SIRT1 levels in anti-FLAG immunoprecipitates in HEK293 cells transfected with FLAG-SIRT6 or an empty vector. (B) Western blot analysis of SIRT6 levels in anti-FLAG … see more(a)转染 FLAG-SIRT6或空载体的 HEK293细胞抗 flag 免疫沉淀物中 SIRT1蛋白的免疫印迹分析。(b) SIRT6蛋白水平的免疫印迹分析

Figure 3—figure supplement 2 图3ー图补充资料2Download asset下载资产Open asset开放资产

SIRT1-SIRT6 interaction.SIRT1-SIRT6相互作用(A) Immunofluorescence analysis of endogenous SIRT1 (Green) and SIRT6 (Red) protein levels. Representative images are shown, captured under a confocal imaging microscope. Scale bar, 10 μm. (B) A … see more内源性 SIRT1(绿色)和 SIRT6(红色)蛋白水平的免疫荧光分析。图为共聚焦成像显微镜拍摄的典型图像。比例尺,10微米。(b) a… 更多信息

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Acetylation level of SIRT6.SIRT6的乙酰化水平The acetylation levels of FLAG-SIRT6 in the presence of NAM (5 mM), TSA (1 μM) or Ex527 (1 μM) were determined by anti-FLAG IP and western blotting using an anti-pan-acetyl lysine antibody in HEK293 … see more在 NAM (5mm)、 TSA (1μM)或 Ex527(1μM)存在下,用抗 flag IP 和西方墨点法测定 FLAG-SIRT6的乙酰化水平,并用 HEK293中的抗泛乙酰赖氨酸抗体进行测定

SIRTs contain a conserved Sir2 domain and flexible N-termini and C-termini. To locate the exact SIRT6 domains that interact with SIRT1, we deleted the N-terminus and C-terminus, as previously reported (Tennen et al., 2010Figure 3—figure supplement 2B,C). Western blotting analysis showed that the interaction between SIRT6 and SIRT1 was lost if the N-terminus or C-terminus of SIRT6 was deleted (Figure 3G). As the C-terminus contains the nuclear location signal (Tennen et al., 2010), we speculate that its depletion may restrict SIRT6 in cytoplasm, thus preventing the interaction between SIRT1 and SIRT6. Thus, the data indicate that SIRT6 physically interacts with SIRT1, most likely through the N-terminus of SIRT6.

SIRTs 包含一个保守的 Sir2结构域和灵活的 N-termini 和 C-termini。为了准确定位与 SIRT1相互作用的 SIRT6结构域,我们删除了之前报道的 n 端和 c 端结构域(Tennen 等人,2010; 图3ー图2B,c)。西方墨点法分析显示,如果删除 SIRT6的 n 端或 c 端,SIRT6和 SIRT1之间的相互作用就会消失(图3 g)。由于 c 末端包含核定位信号(Tennen 等人,2010) ,我们推测它的缺失可能会限制细胞质中的 SIRT6,从而阻止 SIRT1和 SIRT6之间的相互作用。因此,数据表明,SIRT6相互作用(分子生物学) SIRT1,最有可能通过 n 端的 SIRT6。

SIRT1 deacetylates SIRT6 at K33

SIRT1在 K33位脱乙酰化 SIRT6

We next examined whether SIRT1 deacetylates SIRT6 via the direct interaction that we identified above. Overexpression of SIRT1 but not of other sirtuins inhibited FLAG-SIRT6 acetylation (Figure 4A). Conversely, knocking down SIRT1significantly upregulated endogenous SIRT6 acetylation in HEK293 cells (Figure 4B). Furthermore, the SIRT6 acetylation level decreased in the presence of ectopic SIRT1 but not in the presence of its catalytic mutant SIRT1-H363Y (Figure 4C), despite the two proteins still showing a physical interaction, suggesting that SIRT6 is likely a deacetylation target of SIRT1. To test our hypothesis, we established an in vitro deacetylation assay. We eluted recombinant FLAG-SIRT6 with a FLAG peptide from HEK293 cell lysate. We found that SIRT1 deacetylated SIRT6 in the presence of NAD+, while NAM inhibited this process (Figure 4D,E). The deacetylase-inactive SIRT1-H363Y was unable to deacetylate SIRT6.

我们接下来研究 SIRT1是否通过我们上面确定的直接相互作用去乙酰化 SIRT6。过量表达 SIRT1而不表达其他 sirtuins 抑制 FLAG-SIRT6乙酰化(图4A)。相反,敲除 SIRT1显著上调 HEK293细胞内源性 SIRT6乙酰化(图4B)。此外,SIRT6的乙酰化水平在异位 SIRT1的存在下降,但在其催化突变体 SIRT1-h363y 的存在下没有下降(图4C) ,尽管这两个蛋白仍然显示物理相互作用,提示 SIRT6可能是 SIRT1的脱乙酰化目标。为了验证我们的假设,我们建立了体外脱乙酰化实验。我们利用 HEK293细胞裂解液中的 FLAG 多肽洗脱重组 FLAG-SIRT6。我们发现 SIRT1在 NAD + 存在下脱乙酰化 SIRT6,而 NAM 抑制了这一过程(图4D,e)。脱乙酰酶非活性的 SIRT1-H363Y 不能脱乙酰化 SIRT6。Figure 4 图4 with 1 supplement 还有一种补充剂Download asset下载资产Open asset开放资产

SIRT1 deacetylates SIRT6 at K33.SIRT1在 K33位脱乙酰化 SIRT6(A) The acetylation level of FLAG-SIRT6 in HEK293 cells ectopically expressing SIRT1-5 and SIRT7. (B) The acetylation level of endogenous SIRT6 in HEK293 cells treated si-SIRT1 or scramble (Scram) … see more(a)外源性表达 SIRT1-5和 SIRT7的 HEK293细胞中 FLAG-SIRT6的乙酰化水平。(b)经 si-SIRT1或 scramble (Scram)处理的 HEK293细胞内源性 SIRT6的乙酰化水平

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Acetylation levels of SIRTs.乙酰化水平(A) SIRT6 K143/145R acetylation levels in anti-FLAG IPs derived from HEK293 cells, was determined by western blotting with a pan anti-acetyl lysine antibody. (B) FLAG-SIRT1 acetylation levels in … see more用泛抗乙酰赖氨酸抗体测定 HEK293细胞来源的抗 flag IPs 的 SIRT6 K143/145R 乙酰化水平。(b) FLAG-SIRT1乙酰化水平见… 更多

As SIRT1 might interact with the SIRT6 N-terminus, we hypothesized that it might also deacetylate K33ac. While the acetylation level of SIRT6 was increased in SIRT1/ HEK293 cells, that of K33R was hardly affected (Figure 4F). Additionally, the acetylation level of SIRT6 K33R was minimally changed upon SIRT1 overexpression (Figure 4G), whereas that of K143/145R was downregulated upon ectopic SIRT1 (Figure 4—figure supplement 1A). These data support that K33ac is a target of SIRT1. By contrast, the SIRT1 acetylation level was relatively unaffected upon SIRT6 overexpression (Figure 4—figure supplement 1B). To further validate these findings, we synthesized a K33ac-containing peptide and found that it effectively blocked the in vitro binding of SIRT6 to SIRT1 (Figure 4H). Of note, the GST pulldown assay suggested that the N-terminus rather than the C-terminus of SIRT6 was responsible for its interaction with SIRT1. Together, these data suggest that SIRT1 deacetylates SIRT6 at K33.

由于 SIRT1可能与 SIRT6的 n 端相互作用,我们推测它也可能与 K33ac 脱乙酰基有关。SIRT1-/-HEK293细胞 SIRT6的乙酰化水平升高,而 K33R 的乙酰化水平几乎不受影响(图4F)。此外,SIRT1过表达时 SIRT6 K33R 的乙酰化水平变化不大(图4G) ,而 K143/145R 在异位 SIRT1时乙酰化水平下调(图4ー图1A)。这些数据支持 K33ac 是 SIRT1的目标。相反,SIRT1乙酰化水平在 SIRT6过表达时相对较低(图4ー图1B)。为了进一步验证这些发现,我们合成了一个含有 k33ac 的肽,并发现它有效地阻断了 SIRT6与 SIRT1的体外结合(图4H)。值得注意的是,GST 下拉实验表明 SIRT6与 SIRT1的相互作用是由 n 端而不是 c 端引起的。总之,这些数据表明,SIRT1在 K33脱乙酰化 SIRT6。

γH2AX ensures SIRT6 retention surrounding DSBs

γh2ax 确保 SIRT6保持在 DSBs 周围

γH2AX is dispensable for the initial DSB recognition but serves as a platform for recruiting DDR factors (Celeste et al., 2003). Because SIRT6 is enriched at DNA breaks, we next asked whether γH2AX is involved in SIRT6 recruitment. We thus performed a co-IP of endogenous SIRT6 in cells treated with or without camptothecin (CPT). Interestingly, H2AX and γH2AX were detected in the anti-SIRT6 precipitates only when the cells were treated with CPT (Figure 5A,B). We also performed an in vitro pulldown assay with a biotin-labeled C-terminal γH2AX peptide (biotin-γH2AXp) or H2AX peptide (biotin-H2AXp). Consistently, GST-SIRT6 recognized the γH2AX peptide but not the H2AX peptide (Figure 5C). To identify the interacting domain, we purified various GST-SIRT6 truncation mutants. Peptide pulldown assay revealed that the N-terminus truncation was enough to abolish SIRT6 binding to γH2AX peptide, while the C-terminus truncation had a minimal effect (Figure 5D). We then investigated whether SIRT1-mediated deacetylation contributes to SIRT6 binding to γH2AX. Here, the K33R mutant efficiently bound to γH2AX to a similar extent as WT SIRT6, but the binding was abolished in the case of K33Q (Figure 5E).

γh2ax 对于 DSB 的初始识别是可有可无的,但它是招募 DDR 因子的一个平台(Celeste 等人,2003)。因为 SIRT6在 DNA 断裂时富集,我们接下来要问 γh2ax 是否参与了 SIRT6的补充。因此,我们在接受或不接受喜树碱(CPT)处理的细胞中进行了内源性 SIRT6的共同激发态。有趣的是,只有当用 CPT 处理细胞时,抗 sirt6沉淀物中才检测到 H2AX 和 γH2AX (图5A,b)。我们还用生物素标记的 c 末端 γH2AX 多肽(biotin-γH2AXp)或 H2AX 多肽(biotin-H2AXp)进行了体外下拉实验。GST-SIRT6识别的是 γH2AX 多肽,而不是 H2AX 多肽(图5C)。为了鉴定相互作用结构域,我们纯化了多个 GST-SIRT6的截短突变体。肽段下拉实验表明,n 端截断足以阻断与 γh2ax 肽的 SIRT6结合,而 c 端截断效果最小(图5D)。然后我们研究了 sirt1介导的脱乙酰基是否参与了 SIRT6与 γh2ax 的结合。在这里,K33R 突变体与 γh2ax 的结合程度与 WT SIRT6相似,但是在 K33Q 的情况下结合被消除了(图5E)。Figure 5 图5 with 1 supplement 还有一种补充剂Download asset下载资产Open asset开放资产

γH2AX is required for the chromatin retention of SIRT6.SIRT6的染色质保留需要 γh2ax 参与(A,B) Representative western blots showing H2AX (A) and γ-H2AX (B) levels in anti-SIRT6 immunoprecipitates from HEK293 cells treated with or without 1 μM camptothecin (CPT). The IgG control … see more(a,b)经喜树碱(CPT)处理或不处理的 HEK293细胞表达 H2AX (a)和 γ-H2AX (b)抗 sirt6免疫沉淀物。免疫球蛋白抗体控制… 详见

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SIRT6 recruitment in cells treated with caffeine.咖啡因对细胞 SIRT6的补充作用(A) Laser MicroPoint analysis of SIRT6 recruitment in MEFs treated with caffeine (5 mM) 1 hr. Scale bar, 10 μm. (B) The relative intensity was calculated in Fiji (Image J). The data represent the … see more(a)咖啡因(5mm)1hr 对大鼠 MEFs SIRT6招募的激光微点分析。比例尺,10微米。(b)在斐济计算相对强度(图 j)。这些数据代表了… 更多

To investigate the functional relevance of this SIRT6–γH2AX interaction, we applied laser-induced DNA damage in MEFs lacking H2ax and then tracked the distribution of SIRT6 by immunofluorescence microscopy. GFP-SIRT6 was immediately recruited to DNA lesions in H2ax+/+ and H2ax−/− MEFs (Figure 5F,G), implying that H2AX is dispensable for initial SIRT6 recruitment. Interestingly, GFP-SIRT6 diminished from DNA lesions at 10 min after laser treatment in H2ax–/– MEFs but persisted in H2ax+/+ cells. H2AX is rapidly phosphorylated at serine 139 in response to DSBs (Rogakou et al., 1998). When we re-introduced H2AX WT, S139A and S139D into H2ax–/– MEFs, SIRT6 retention was restored in WT and S139D-re-expressing cells but not in S139A-re-expressing cells (Figure 5H,I). Moreover, we used caffeine, an ATM/ATR kinase inhibitor, to treat cells and did SIRT6 recruitment assay. The data showed that caffeine inhibited the level of γH2AX and subsequent retention of SIRT6 at DSBs after laser-induced DNA damage in MEFs (Figure 5—figure supplement 1). Together, these data indicate that SIRT6 recognizes γH2AX surrounding DSBs and that this recognition is enhanced by SIRT1-mediated deacetylation.

为了研究 SIRT6-γH2ax 相互作用的功能相关性,我们在缺乏 H2ax 的 MEFs 中应用激光诱导 DNA 损伤,然后用免疫荧光显微镜跟踪 SIRT6的分布。GFP-SIRT6被立即招募到 H2AX +/+ 和 H2AX-/-MEFs (图5F,g)的 DNA 损伤中,这意味着 H2AX 对于初始 SIRT6招募是可有可无的。有趣的是,在 H2ax-/-MEFs 激光处理后10分钟,GFP-SIRT6从 DNA 损伤中消失,但在 H2ax +/+ 细胞中仍然存在。针对 DSBs,H2AX 在丝氨酸139处迅速被磷酸化(rogaykou 等人,1998)。当我们将 H2AX WT、 S139A 和 S139D 重新导入 H2AX-/-MEFs 时,SIRT6在 WT 和 S139D–re 表达细胞中的保留得到恢复,而在 S139A–re 表达细胞中则没有恢复(图5H,i)。此外,我们还用 ATM/ATR 激酶抑制剂咖啡因对细胞进行治疗,并进行 SIRT6招募试验。结果表明,咖啡因可抑制激光诱导 MEFs DNA 损伤后 γh2ax 水平和 SIRT6在 DSBs 上的滞留(图5ー图1)。总之,这些数据表明,SIRT6识别 γh2ax 周围的 DSBs,这种识别是通过 sirt1介导的脱乙酰化来提高的。

SIRT1 and SIRT6 cooperatively promote DNA repair

SIRT1和 SIRT6协同促进 DNA 修复

The physical interaction between SIRT1 and SIRT6 prompted us to further investigate whether SIRT1 and SIRT6 cooperatively modulate the DDR and DNA repair. To do so, we combined the DR-GFP reporter system with a ChIP-PCR analysis. First, we found that FLAG-SIRT6 recruitment to the DSB vicinity was significantly reduced when SIRT1 was silenced by siRNA in HEK293 cells (Figure 6A,B). Then we monitored the dynamic recruitment of GFP-SIRT6 upon laser-induced DNA damage using a MicroPoint system. GFP-SIRT6 was rapidly recruited to DSBs in WT cells, but this process was largely deferred in Sirt1–/– MEFs (Figure 6C,D), suggesting an indispensable role of SIRT1 in the initial recruitment of SIRT6 to DSBs. By contrast, SIRT1 recruitment to DSBs was relatively unaffected by SIRT6 downregulation, as determined by the DR-GFP reporter system (Figure 6E,F) and the MicroPoint system (Figure 6G,H).

SIRT1和 SIRT6之间的物理相互作用促使我们进一步研究 SIRT1和 SIRT6是否协同调节 DDR 和 DNA 修复。为此,我们将 DR-GFP 报告系统与 ChIP-PCR 分析相结合。首先,我们发现,当 HEK293细胞中的 siRNA 沉默 SIRT1时,FLAG-SIRT6对 DSB 附近的补充显著减少(图6 a,b)。然后,我们利用微点系统监测 GFP-SIRT6在激光诱导 DNA 损伤时的动态补充。GFP-SIRT6在野生动物小组的 dsb 中迅速招募,但 SIRT1-/-MEFs (图6C,d)基本上推迟了这一进程,这表明 SIRT1在 SIRT6初期招募到 DSBs 中发挥了不可或缺的作用。相比之下,SIRT1招募到 dsb 相对不受 SIRT6下调的影响,这是由 DR-GFP 报告系统(图6E,f)和 MicroPoint 系统(图6G,h)确定的。Figure 6 图6Download asset下载资产Open asset开放资产

SIRT1 facilitates SIRT6 recruitment to chromatin during the DDR.SIRT1促进复员时 SIRT6招募染色质(A,B) ChIP-qPCR analysis of the SIRT6 levels in the vicinity of a DSB in cells treated with a SIRT1 siRNA (si-SIRT1) or a scrambled negative control (NC). The western blots show the FLAG-SIRT6 and … see moreSIRT1 siRNA (si-SIRT1)和错配阴性对照(NC)对 DSB 附近细胞 SIRT6水平的(a,b) ChIP-qPCR 分析。西部斑点显示 FLAG-SIRT6和… 更多信息

SIRT6 rescues DNA repair defects caused by SIRT1 deficiency

SIRT6对 SIRT1缺陷引起的 DNA 修复缺陷的修复作用

In our final set of assays, we wanted to determine the function of SIRT6 deacetylation in DNA repair. We found that the interaction of SIRT1 and SIRT6 was enhanced upon DNA damage (Figure 7A). In addition, SIRT6 acetylation was significantly decreased upon CPT treatment, but that the effects of CPT were abolished in the presence of SIRT6 K33R or in the absence of SIRT1 (Figure 7B,C). These data imply that SIRT6 is deacetylated by SIRT1 upon DNA damage. We then examined the effects of the SIRT6 mutants on DNA repair by comet assay, which assesses the DNA repair ability at the single cell level. We overexpressed SIRT6 K33R or K33Q in SIRT6–/– cells and then examined the DNA repair efficacy. Here, overexpression of SIRT6 significantly enhanced DNA repair efficacy upon CPT treatment, while K33Q or H133Y lost the ability. By contrast, the overexpression of SIRT6 K33R promoted DNA repair to an extent comparable to WT (Figure 7D,E and Figure 7—figure supplement 1A). An HR assay showed that SIRT6 WT and K33R but neither K33Q nor H133Y enhanced HR capacity (Figure 7F and Figure 7—figure supplement 1B). We further assessed cell viability of SIRT6–/– HEK293 cells reconstituted with SIRT6 mutants using an MTS assay. The data showed that the viability of cells transfected with SIRT6 WT or K33R was much higher than those transfected with empty vector, SIRT6 K33Q or H133Y after CPT or IR treatment (Figure 7—figure supplement 2). In HeLa cells, overexpression of SIRT6 K33Q also inhibited the colony-forming capacity upon CPT or IR treatment compared to SIRT6 WT and K33R (Figure 7—figure supplement 3). In addition, less γH2AX foci was noticed in HeLa cells transfected ectopic SIRT6 WT or K33R compared to K33Q or H133Y at 8 hr after IR (Figure 7—figure supplement 4). These data implicate that SIRT6 deacetylation at K33 is indispensable for DNA repair.

在我们的最后一组实验中,我们想要确定 SIRT6去乙酰化在 DNA 修复中的作用。我们发现 SIRT1和 SIRT6的相互作用在 DNA 损伤时增强(图7A)。此外,经 CPT 处理后,SIRT6乙酰化水平显著降低,但在 SIRT6 K33R 存在或 SIRT1缺失的情况下,CPT 效应消失(图7B,c)。这些数据表明,SIRT6在 DNA 损伤时被 SIRT1去乙酰化。然后用彗星实验检测了 SIRT6突变体对 DNA 修复的影响,从单细胞水平评价了 DNA 修复能力。我们在 SIRT6-/-细胞中高表达了 SIRT6 K33R 或 K33Q,并检测了其 DNA 修复效果。这里,SIRT6的过度表达显著提高了 CPT 治疗后的 DNA 修复效果,而 K33Q 或 H133Y 则丧失了修复能力。相比之下,SIRT6 K33R 的过度表达促进了 DNA 修复,其程度可与 WT 相媲美(图7D、 e 和图7ー图补充1A)。HR 测定表明,SIRT6 WT 和 K33R 均能提高 HR 能力,但 K33Q 和 H133Y 均不能提高 HR 能力(图7F 和图7ー图1B)。我们进一步评估细胞活力的 SIRT6-/-HEK293细胞重组与 SIRT6突变体使用 MTS 分析。结果表明,经 CPT 或 IR 处理后,转染 SIRT6 WT 或 K33R 的细胞存活率明显高于空载体 SIRT6 K33Q 或 H133Y 的细胞存活率(图7ー图2)。在 HeLa 细胞中,与 SIRT6 WT 和 K33R 相比,SIRT6 K33Q 的过表达也抑制了 CPT 和 IR 处理后的集落形成能力(图7ー图3)。此外,在 IR 后8小时,转染异位 SIRT6 WT 或 K33R 的 HeLa 细胞中 γh2ax 病灶较 K33Q 或 H133Y 少(图7ー图4)。这些数据表明,在 K33位点的 SIRT6脱乙酰基是 DNA 修复不可缺少的基因。Figure 7 图7 with 5 supplements 有5种补充剂Download asset下载资产Open asset开放资产

SIRT6 rescues DNA repair defects caused by a SIRT1 deficiency.SIRT6修复 SIRT1缺陷引起的 DNA 修复缺陷(A) Co-IP and western blot analysis of the interaction of FLAG-SIRT1 and SIRT6 in HEK293 cells overexpressing FLAG-SIRT1 and treated with CPT (1 μM) for 1 hr. (B–C) The acetylation level of SIRT6 WT … see more(a)过表达 FLAG-SIRT1的 HEK293细胞经 CPT (1μM)处理1h 后,用 Co-IP 和 western blot 分析 FLAG-SIRT1与 SIRT6的相互作用。(b-c) SIRT6 WT 的乙酰化水平..

Figure 7—figure supplement 5 图7ー附图5Download asset下载资产Open asset开放资产

SIRTs levels in SIRT1–/–cells.SIRT1-/-细胞中 SIRTs 水平的研究(A) Western blots showing SIRT6 and SIRT1 levels in SIRT1–/–cells transfected with SIRT6 WT, K33R, K33Q, H133Y or SIRT1 constructs. (B) Western blots showing SIRT6 and SIRT1 levels in SIRT1–/–… see more(a)转染 SIRT6 WT、 K33R、 K33Q、 H133Y 或 SIRT1基因的 SIRT1-/-细胞表达 SIRT6和 SIRT1蛋白的蛋白印迹。(b)西部杂交结果显示 SIRT6和 SIRT1在 SIRT1-/… 见更多

Figure 7—figure supplement 4 图7ー图补充4Download asset下载资产Open asset开放资产

γH2AX foci in HeLa cells.HeLa 细胞中的 γh2ax 焦点(A) Immunofluorescence staining of γH2AX foci in HeLa cells expressing ectopic FLAG-SIRT6 WT, K33R, K33Q or H133Y at 8 hr after radiation. Scale bar, 10 μm. (B) Quantification of γH2AX foci-positive … see more(a)照射后8小时,γh2ax 免疫荧光染色表达异位的 FLAG-SIRT6 WT、 K33R、 K33Q 或 H133Y。比例尺,10微米。(b) γh2ax 焦点阳性的量化… 见更多

Figure 7—figure supplement 3 图7ー图补充3Download asset下载资产Open asset开放资产

Colony-forming ability of HeLa cells.HeLa 细胞的集落形成能力(A) Western blots showing SIRT6 protein levels in HeLa cells stably transfected with FLAG-SIRT6 WT, K33R, K33Q constructs. (B) Colony-forming assay in HeLa cells ectopically expressing FLAG-SIRT6, … see more(a) flag-SIRT6 WT,K33R,K33Q 基因转染 HeLa 细胞后,蛋白质印迹显示 SIRT6蛋白水平稳定。(b)在 HeLa 细胞中体外表达 FLAG-SIRT6的集落形成实验,… 更多

Figure 7—figure supplement 2 图7ー附图2Download asset下载资产Open asset开放资产

Cell viability assay in SIRT6–/–cells.SIRT6-/-细胞活性测定(A) Western blots showing SIRT6 protein levels in SIRT6–/–HEK293 cells transfected with FLAG-SIRT6 WT, K33R, K33Q or H133Y constructs. (B) Cell viability of SIRT6–/–HEK293 cells expressing ectopic … see more(a)转染 flag-SIRT6 WT、 K33R、 K33Q 或 H133Y 基因的 SIRT6-/-HEK293细胞的蛋白水平。(b) SIRT6-/-HEK293细胞异位表达的细胞活性

Figure 7—figure supplement 1 图7ー附图1Download asset下载资产Open asset开放资产

SIRT6 levels in SIRT6–/–cells.SIRT6在 SIRT6-/-细胞中的表达(A) Western blots showing SIRT6 protein levels in SIRT6–/–HEK293 cells transfected with FLAG-SIRT6 WT, K33R, K33Q and H133Y constructs. Related to Figure 7D,E. (B) Western blots showing SIRT6 … see more(a)转染 flag-SIRT6 WT、 K33R、 K33Q 和 H133Y 基因的 SIRT6-/-HEK293细胞的蛋白水平。与图7D 相关,e (b)显示 SIRT6的西部 blots… 参见更多

SIRT1 regulates DNA repair (Wang et al., 2008). To elucidate the synergistic effects of SIRTs in DNA repair, we examined whether SIRT6 hyper-acetylation is responsible for the defective DNA repair capacity seen in SIRT1–/– HEK293 cells. SIRT6 WT, K33R and SIRT1 overexpression rescued the defective DNA repair imposed by the SIRT1 deficiency, while SIRT6 K33Q and H133Y had minimal rescue effect (Figure 7G,H and Figure 7—figure supplement 5A). Notably, both SIRT6 WT and K33R had similar function in rescuing the DNA repair defect in SIRT1 KO cells, suggesting that overexpressed exogenous SIRT6 WT might not be effectively acetylated. Further, the HR assay showed that SIRT6 WT and K33R, but neither K33Q nor H133Y rescued the HR defect caused by SIRT1 deficiency (Figure 7I and Figure 7—figure supplement 5B).

SIRT1管理 DNA 修复(Wang et al. ,2008)。为了阐明 SIRTs 在 DNA 修复中的协同作用,我们研究了 SIRT6高乙酰化是否与 SIRT1-/-HEK293细胞 DNA 修复能力的缺陷有关。SIRT6 WT、 K33R 和 SIRT1过表达能够修复 SIRT1缺陷引起的 DNA 修复缺陷,而 SIRT6 K33Q 和 H133Y 的修复效果最小(图7G、 h 和图7ー图5A)。值得注意的是,SIRT6 WT 和 K33R 在挽救 SIRT1 KO 细胞 DNA 修复缺陷方面具有相似的功能,提示过度表达的外源性 SIRT6 WT 可能不能有效地被乙酰化。此外,HR 分析显示 SIRT6 WT 和 K33R,但 K33Q 和 H133Y 均不能挽救 SIRT1缺陷引起的 HR 缺陷(图7I 和图7ー图5B)。

Altogether, these data implicate a synergistic action between SIRT1 and SIRT6 in regulating the DDR and DNA repair. We thus propose a model by which SIRT6 is deacetylated by SIRT1 at K33, thus promoting its polymerization and recognition of DSBs; SIRT6 that is deacetylated at K33 anchors to γH2AX, allowing retention on the chromatin flanking the DSBs and subsequent remodeling via deacetylating H3K9ac and H3K56ac (Figure 8).

总之,这些数据暗示了 SIRT1和 SIRT6在调节 DDR 和 DNA 修复中的协同作用。因此,我们提出了一个 SIRT6在 K33上被 SIRT1去乙酰化的模型,从而促进了 DSBs 的聚合和识别; SIRT6在 K33上被去乙酰锚定到 γh2ax 上,允许保留在 DSBs 的染色质侧翼,并通过去乙酰化 H3K9ac 和 H3K56ac 进行重塑(图8)。Figure 8 图8Download asset下载资产Open asset开放资产

A working model.一个工作模型(A) SIRT6 is deacetylated by SIRT1 at K33, which promotes SIRT6 polymerization and recognition of DSBs. (B) Beyond DSBs, K33-deacetylated SIRT6 anchors to γH2AX and expands on local chromatin … see more(a) SIRT6在 K33位被 SIRT1脱乙酰,促进了 SIRT6的聚合和双链识别。除了 DSBs,k33脱乙酰基 SIRT6锚定到 γh2ax 并扩展到局部染色质… 更多

Discussion 讨论

The DDR is a highly orchestrated process that is initiated by DNA break-sensing (Ciccia and Elledge, 2010). While the MRN complex (Paull and Lee, 2005), Ku complex (Hu et al., 2012), RPA (Maréchal and Zou, 2015) and PARP1 (Ali et al., 2012Eustermann et al., 2015) are all known to directly recognize DSBs, sirtuins are among the earliest factors to be recruited to DSBs (Dobbin et al., 2013Toiber et al., 2013) and facilitate PARP1 recruitment (Vazquez et al., 2016). Consistent with published data (Pan et al., 2011), we found that SIRT6 oligomerizes and recognizes DSBs via a DSB-binding pocket generated by the N-termini and C-termini of two adjacent molecules. This finding is consistent with another report showing that both the N-termini and C-termini are essential for the chromatin association of SIRT6 . Using a super-resolution fluorescent particle tracking method, Yang et al. recently found that PARP1 binding to DSBs happens earlier than SIRT6 binding (Yang et al., 2018). One possible explanation is that PARP1 is first recruited to DSBs; then, SIRTs are later recruited directly by DSBs and facilitate PARP1 stabilization and expansion in the surrounding region.

DDR 是一个高度协调的过程,由 DNA 断裂感应开始(Ciccia and Elledge,2010)。虽然 MRN 复合体(Paull 和 Lee,2005年)、 Ku 复合体(Hu 等人,2012年)、 RPA (Maréchal 和 Zou,2015年)和 PARP1(Ali 等人,2012年; Eustermann 等人,2015年)都已知可直接识别 DSBs,sirtuin 是 DSBs 招募的最早因素之一(Dobbin 等人,2013年; Toiber 等人,2013年) ,并为 PARP1招募提供便利(Vazquez 等人,2016年)。与已发表的数据一致(Pan 等人,2011年) ,我们发现 SIRT6通过两个相邻分子的 N-termini 和 C-termini 产生的 dsb 结合口袋来寡聚和识别 DSBs。这一发现与另一篇报道相一致,该报道表明 n 端和 c 端对 SIRT6的染色质联系至关重要。通过使用超分辨率的荧光粒子跟踪方法,Yang 等人最近发现 PARP1与 DSBs 的结合比 SIRT6的结合早(Yang 等人,2018)。一个可能的解释是 PARP1首先被招募到 dsb; 然后,sirt 随后被 dsb 直接招募,并促进 PARP1在周围地区的稳定和扩展。

The sirtuin family members share similar functions in the DDR and in DNA repair; upon DNA damage, both SIRT1 and SIRT6 are rapidly mobilized to DSBs (Vazquez et al., 2016Dobbin et al., 2013Toiber et al., 2013). SIRT1 redistributes on chromatin and deacetylates XPA, NBS1 and Ku70 to promote DNA repair (Fang et al., 2016Yuan et al., 2007Fan and Luo, 2010Jeong et al., 2007). Recently, an elegant study demonstrated that PAR recruits SIRT1 and BRG1 to DSB sites and promotes HR efficiency (Chen et al., 2019). Other studies reported that SIRT6 mono-ribosylates PARP1 to enhance its activity (Mao et al., 2011), and SIRT6 facilitates the subsequent recruitment of SNF2H, H2AX and DNA-PKcs (Atsumi et al., 2015McCord et al., 2009Van Meter et al., 2016). Here, we revealed a synergistic action between two nuclear SIRTs in DDR−SIRT1 deacetylates SIRT6 to promote its mobilization to DSBs. A K33R mutant, mimicking the hypo-acetylated SIRT6, can rescue DNA repair defects in SIRT1null cells. Both BRG1 and SNF2H are chromatin remodeling ATPases, responsible for open chromatin architecture. It is reasonable to speculate that these early DDR responding factors like PARP1, SIRT1, SIRT6, SNF2H and BRG1 are quickly and sequentially stimulated by DSBs, wherein they constitute a super complex to potentiate DDR and DNA repair; posttranslational modifications like deacetylation and mono-ADP ribosylation empower the complex to recruit other repair factors more efficiently. Interestingly, SIRT6 phosphorylation at S10 by JNK promotes subsequent recruitment itself and PARP1 upon oxidative stress, also supporting an essential role of the SIRT6 N terminus for DSB-recruitment (Van Meter et al., 2016). Consistent with the cooperative action between SIRT1 and SIRT6, independent studies have revealed an interaction between SIRT1 and SIRT7, showing that SIRT1 recruits SIRT7 to promote cancer cell metastasis (Malik et al., 2015), and that SIRT1 and SIRT7 antagonistically regulate adipogenesis (Fang et al., 2017).

Sirtuin 家族成员在 DDR 和 DNA 修复中具有相似的功能; 在 DNA 损伤时,SIRT1和 SIRT6都会迅速被动员到 DSBs (Vazquez et al. ,2016; Dobbin et al. ,2013; Toiber et al. ,2013)。SIRT1在染色质和去乙酰化物 XPA、 NBS1和 Ku70上重新分布以促进 DNA 修复(Fang et al. ,2016; Yuan et al. ,2007; Fan and Luo,2010; Jeong et al. ,2007)。最近,一项优雅的研究表明 PAR 将 SIRT1和 BRG1招募到 DSB 位点并提高人力资源效率(Chen et al. ,2019)。其他研究报道,SIRT6单核糖基化 PARP1,以提高其活性(Mao 等人,2011年) ,和 SIRT6促进随后补充 SNF2H,H2AX 和 DNA-PKcs (Atsumi 等人,2015; McCord 等人,2009; Van Meter 等人,2016年)。在这里,我们揭示了在 DDR-SIRT1脱乙酰基 SIRT6两个核 sirt 之间的协同作用,以促进其动员到 dsb。一个模仿低乙酰化 SIRT6的 K33R 突变体可以修复 SIRT1缺失细胞的 DNA 修复缺陷。BRG1和 SNF2H 都是染色质重塑 ATPases,负责开放染色质结构。这些早期的 DDR 反应因子如 PARP1、 SIRT1、 SIRT6、 SNF2H 和 BRG1可以被 dsb 快速、顺序地刺激,其中它们构成一个超复合体以增强 DDR 和 DNA 修复,翻译后的修饰如脱乙酰化和单 adp 核糖基化使复合体更有效地吸收其他修复因子。有趣的是,JNK 在 S10位点的 SIRT6磷酸化促进了后续的补充和 PARP1,也支持了 SIRT6 n 末端对 dsb 补充的重要作用(Van Meter et al. 2016)。与 SIRT1和 SIRT6之间的合作作用一致,独立研究揭示了 SIRT1和 SIRT7之间的相互作用,表明 SIRT1招募 SIRT7促进癌细胞转移(Malik 等人,2015) ,而且 SIRT1和 SIRT7拮抗调节脂肪生成(Fang 等人,2017)。

The acetylation levels of H3K9 and H3K56 decrease upon detecting DSBs and then return to basal levels (Tjeertes et al., 2009). SIRT1 and SIRT6 are H3K9ac and H3K56ac deacetylases; both are recruited to DSBs, indicating that SIRT1 and/or SIRT6 might contribute to reducing H3K9ac and H3K56ac levels. Although mechanistically unclear, H3K9ac and H3K56ac levels negatively correlate with γH2AX levels (Tjeertes et al., 2009). In this study, we found that while γH2AX is not required for initial SIRT6 recruitment, it is indispensable for retaining SIRT6 on the local chromatin surrounding DSBs. This finding is consistent with reports that γH2AX is dispensable for initial reorganization of DNA breaks but rather serves as a platform to stabilize DNA repair factors, such as NBS1, 53BP1 and BRCA1. SIRT6 deacetylates H3K9ac and H3K56ac surrounding DSBs, in this way bridging γH2AX to chromatin remodeling. While the in vivo data demonstrated that SIRT6 K33Q deacetylation activity toward histone H3 was compromised, the in vitro deacetylation assay using a synthesized acetyl H3 peptide showed negligible effect. It is speculated that the initial DSB recognition and chromatin retention might potentiate the deacetylase activity of SIRT6 toward local histones, for example H3K9ac and H3K56ac; the impaired DSB recognition and chromatin retention might compromise the deacetylase activity of SIRT6 K33Q on local histone proteins. Putting together the findings provide a scenario as to how γH2AX and histone modifiers coordinate to amplify the DDR.

H3K9和 H3K56的乙酰化水平在检测到 dsb 后下降,然后回到基础水平(Tjeertes 等,2009)。SIRT1和 SIRT6分别是 H3K9ac 和 H3K56ac 脱乙酰酶,都被招募到 DSBs 中,这表明 SIRT1和/或 SIRT6可能有助于降低 H3K9ac 和 H3K56ac 水平。虽然机理上不清楚,H3K9ac 和 H3K56ac 水平与 γh2ax 水平呈负相关(Tjeertes 等人,2009)。在这项研究中,我们发现,虽然 γh2ax 不需要初始 SIRT6招募,它是必不可少的保留 SIRT6对周围的局部染色质 DSBs。这一发现与以下报道一致: γh2ax 对于 DNA 断裂的初始重组是不可或缺的,而是作为稳定 DNA 修复因子的平台,如 NBS1、53BP1和 BRCA1。6去乙酰化 H3K9ac 和 H3K56ac 周围的 DSBs,以这种方式连接 γh2ax 到染色质重塑。虽然体内数据显示 SIRT6 K33Q 对组蛋白 H3的脱乙酰活性受到损害,但体外合成乙酰 H3肽的脱乙酰活性测定显示微弱的影响。推测 DSB 的初始识别和染色质保留可能增强了 SIRT6对 H3K9ac 和 H3K56ac 等组蛋白的去乙酰化酶活性,而 DSB 识别和染色质保留受损可能影响了 SIRT6 K33Q 对局部组蛋白的去乙酰化酶活性。综合这些研究结果,我们可以推测出 γh2ax 和组蛋白修饰剂是如何协同增强 DDR 的。

SIRT6 and SNF2H cooperate to stabilize γH2AX foci (Atsumi et al., 2015). Here we found that γH2AX in-fact anchors SIRT6 to DSBs, providing a positive feedback regulatory loop between SIRT6 and γH2AX. This finding is consistent with reports showing a distinct reduction of γH2AX and an improper DDR in Sirt6–/– and Sirt1–/– cells. Recent work also suggests that an electrostatic force between a negatively charged phosphate group and a positively charged lysine groups is a novel form of protein–protein interaction (Wang et al., 2016). We thus consider it plausible to speculate that (de)acetylation might act as a switch to modulate such an interaction between SIRT6 and γH2AX.

SIRT6和 SNF2H 协同稳定 γh2ax 焦点(Atsumi 等人,2015)。在这里,我们发现 γh2ax 实际上将 SIRT6锚定到 dsb,在 SIRT6和 γh2ax 之间提供了一个正反馈调节环。这一发现与报告显示的 γh2ax 明显减少和 Sirt6-/-和 Sirt1-/-细胞中不适当的 DDR 一致。最近的研究也表明,带负电荷的磷酸基团和带正电荷的赖氨酸基团之间的静电力是蛋白质-蛋白质相互作用的一种新形式(Wang 等人,2016)。因此,我们推测(去)乙酰化可能是调节 SIRT6和 γh2ax 之间相互作用的开关。

Known as longevity-associated genes, SIRT6 and SIRT1 are redundant in DNA repair but not replaceable. In this study, we have identified that SIRT6 directly binds to DNA breaks and have elucidated a physical and functional interaction between SIRT6 and SIRT1. SIRT6 rescues DNA repair defects imposed by SIRT1 deficiency. Overall, these data highlight a synergistic action of nuclear SIRTs in the spatiotemporal regulation of the DDR and DNA repair.

被称为长寿相关基因,SIRT6和 SIRT1在 DNA 修复中是冗余的,但不可替换。在这项研究中,我们确定了 SIRT6直接结合 DNA 断裂,并阐明了 SIRT6和 SIRT1之间的物理和功能相互作用。SIRT6对 SIRT1缺陷引起的 DNA 修复缺陷的修复作用。总的来说,这些数据突出了核 SIRTs 在 DDR 和 DNA 修复的时空调节中的协同作用。Materials and methods 材料和方法Key resources table 关键资源表

Reagent type 试剂类型
(species) or resource (物种)或资源
Designation 名称Source or reference 来源或参考资料Identifiers 标识符Additional information 补充资料
Gene ( 吉恩(Homo sapiens 智人)SIRT6National Center for Biotechnology Information 美国国家生物技术信息中心Gene ID: 51548 基因 ID: 51548
Gene ( 吉恩(Homo sapiens 智人)SIRT1National Center for Biotechnology Information 美国国家生物技术信息中心Gene ID: 23411 基因 ID: 23411
Gene ( 吉恩(Mus musculus小家鼠)H2axNational Center for Biotechnology Information 美国国家生物技术信息中心Gene ID: 15270 基因 ID: 15270
Gene ( 吉恩(Homo sapiens 智人)H2AXNational Center for Biotechnology Information 美国国家生物技术信息中心Gene ID: 3014 基因 ID: 3014
Cell line ( 细胞系(Homo sapiens 智人)HEK293ATCC 交通管制委员会ATCC CRL-1573
Cell line ( 细胞系(Homo sapiens 智人)HeLa 女名女子名ATCC 交通管制委员会ATCC CRM-CCL-2
Cell line 细胞系
(Mus musculus小家鼠)
MEF 微生物生长因子Dr Linyu Lu (Zhejiang University, China) 陆博士(浙江大学,中国)
Cell line ( 细胞系(Mus musculus小家鼠)H2ax-/- MEF 微生物生长因子Dr Linyu Lu (Zhejiang 陆博士(浙江)
University, China) 大学,中国)
Antibody 抗体SIRT6 (rabbit, polyclonal) SIRT6(兔,多克隆)Abcam (Cambridge, UK) Abcam (剑桥,英国)Cat# ab62738, RRID: 62738,RRID:AB_956299956299Applications: WB; Dilution: 1:1000;Immunofluorescence; Dilution:1:100 用途: WB; 稀释比例: 1:1000; 免疫荧光; 稀释比例: 1:100
Antibody 抗体SIRT1 (mouse, monoclonal) 小鼠,单克隆)Cell Signaling Technology 细胞信号技术Cat# 8469, RRID: 8469,RRID:AB_1099947010999470Applications: WB;Dilution:1:1000;Immunofluorescence; Dilution:1:100 用途: WB; 稀释比例: 1:1000; 免疫荧光; 稀释比例: 1:100
Antibody 抗体FLAG (mouse, monoclonal) FLAG (鼠标,单克隆)Sigma-AldrichCat# F1804; RRID: 1804; RRID:AB_262044262044Applications: WB; Dilution: 1:1000; Chromatin immunoprecipitation 用途: WB; 稀释: 1:1000; 染色质免疫沉淀
Antibody 抗体HA (mouse, monoclonal) HA (鼠,单克隆)Sigma-AldrichCat# H3663; RRID: 3663; RRID:AB_262051262051Applications: WB; Dilution: 1:1000 用途: WB; 稀释: 1:1000
Antibody 抗体GST(mouse, monoclonal) GST (鼠,单克隆)Cell Signaling细胞信号
Technology 技术
Cat# 2624, RRID: 2624,RRID:AB_21898752189875Applications: WB; Dilution: 1:1000 用途: WB; 稀释: 1:1000
Antibody 抗体γH2AX 
(rabbit, monoclonal) (兔子,单克隆)
Abcam (Cambridge, UK) Abcam (剑桥,英国)Cat# ab81299; RRID: 81299; RRID:AB_16405641640564Applications: WB; Dilution: 1:1000 用途: WB; 稀释: 1:1000
Antibody 抗体H3K9ac (rabbit, polyclonal) H3K9ac (兔,多克隆)EMD Millipore 电磁脉冲微孔Cat# 07–352; RRID: 07-352; RRID:AB_310544310544Applications: WB; Dilution: 1:1000 用途: WB; 稀释: 1:1000
Antibody 抗体H3K56ac(Rabbit, Polyclonal) H3K56ac (兔,多克隆)EMD Millipore 电磁脉冲微孔Cat# 07–677, RRID: 07-677号猫,RRID:AB_390167390167Applications: WB; Dilution: 1:1000 用途: WB; 稀释: 1:1000
Antibody 抗体acetyl Lysine 乙酰赖氨酸
(Rabbit, Polyclonal) (兔子,多克隆)
Abcam (Cambridge, UK) Abcam (剑桥,英国)Cat# ab80178, RRID: 80178,RRID:AB_16406741640674Applications: WB; Dilution: 1:1000 用途: WB; 稀释: 1:1000
Transfected construct转染构建体
(Homo sapiens 智人)
pDR-GFPAddgene (Cambridge, MA) Addgene (剑桥,麻省)RRID: 返回文章页面 RRID:Addgene_2647526475
Commercial assay or kit 商业化验或试剂盒CycLex SIRT6 Deacetylase Fluorometric Assay Kit CycLex SIRT6脱乙酰酶荧光测定试剂盒MBL life science MBL 生命科学CY-1156V2
Chemical compound, drug 化合物、药物Ex527Sigma-AldrichE7034
Chemical compound, drug 化合物、药物Trichostatin A 曲古菌素 aSigma-AldrichT1952
Chemical compound, drug 化合物、药物Nicotinamide 烟酰胺Sigma-AldrichN3376
Chemical compound, drug 化合物、药物Camptothecin喜树碱Sigma-AldrichC9911
Software, algorithm 软件,算法GraphPad Prism 石墨平板棱镜GraphPadRRID: 返回文章页面 RRID:SCR_002798002798

Cell lines

细胞系

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HEK293 (CRL-1573) cells and HeLa (CCL-2) cells were ordered from ATCC. H2ax WT and KO mouse embryonic fibroblasts (MEFs) were provided as a kind gift from Dr Linyu Lu (Zhejiang University, China). The cell lines were authenticated by short tandem repeat (STR) profile analysis and genotyping and were mycoplasma free. Cells were routinely cultured in Gibco High Glucose DMEM (Life Technologies, USA) with 10% fetal bovine serum (FBS), 100 U/ml penicillin and streptomycin (P/S) at 37°C in 5% CO2 and atmospheric oxygen conditions.

从 ATCC 中分离出 HEK293(CRL-1573)细胞和 HeLa (CCL-2)细胞。H2ax WT 和 KO 小鼠胚胎成纤维细胞(MEFs)作为礼物由 Linyu Lu 博士(中国浙江大学)提供。这些细胞经过微卫星分子标记(STR)分析和基因分型鉴定无支原体。用10% 胎牛血清(FBS)、100u/ml 青霉素和链霉素(P/S) ,在5% CO2和大气氧气条件下,在 Gibco 高糖 DMEM (Life Technologies,USA)中常规培养细胞。

Oligos and plasmids

Oligos 和质粒

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The following oligos (Genewiz) were used for RNA interference:

下面这些橄榄石(Genewiz)被用于 RNA干扰:

  • siSIRT6, 5’-AAGAAUGUGCCAAGUGUAAGA-3’;siSIRT6,5’-aagaaugugcaaguaaga-3’ ;
  • siSIRT1, 5’-ACUUUGCUGUAACCCUGUA-3’.siSIRT1,5’-acuugcuaccugua-3’。

The following primers were used for ChIP qPCR:

以下是用于芯片 qPCR 的引物:

  • I-SceI- 2 k-F, 5’-GCCCATATATGGAGTTCCGC-3’;I-SceI-2 k-F,5’-gcccatatagttccgc-3’ ;
  • I-SceI-2k-R, 5’-GGGCCATTTACCGTCATTG-3’;I-SceI-2k-R,5’-gggcctattctcattg-3’ ;
  • I-SceI-5k-F, 5’-GTTGCCGGGAAGCTAGAGTAAGTA-3’;I-SceI-5k-F,5’-gttgccgaagtagtagtaagta-3’ ;
  • I-SceI-5k-R, 5’-TTGGGAACCGGAGCTGAATGAA-3’.I-se-5k-r,5’-ttgggaacggagctgaatgaa-3’。

The following gRNA sequences were used for CRISPR/Cas9 gene editing:

下面的 gRNA 序列用于 CRISPR/Cas9基因编辑:

  • Hu Sirt6: gRNA-F, 5’-CACCGGCTGTCGCCGTACGCGGACA-3’;胡士泰6: gRNA-F,5’-caccggctgtcgccgtcgtcggcgcaca-3’ ;
  • gRNA-R, 5’-AAACTGTCCGCGTACGGCGACAGCC-3’.gRNA-R,5’-aaactgtcccgctacggcgcc-3’。
  • Hu Sirt1: gRNA-F, 5’-CACCGATAGCAAGCGGTTCATCAGC-3’胡志成1: gRNA-F,5’-caccgatagggtcatcatcagc-3’

Human SIRT6 was cloned into pCDNA3.1 with a FLAG tag (Invitrogen, USA); a 3 × FLAG-SIRT1 and DR-GFP plasmids were obtained from Addgene. SIRT6ΔC and ΔN were amplified with specific primers and cloned into pKH3HA (Addgene) and pGex vectors (GE Healthcare Life Sciences). The SIRT6 KR, KQ and HY mutants were obtained by converting SIRT6 lysine 33 to arginine (KR), or to glutamine (KQ) and SIRT6 133 histidine to tyrosine (HY) via site-directed mutagenesis, as described below.

将人 SIRT6基因克隆到含 FLAG 标签的 pCDNA3.1中,从 Addgene 获得了3 × FLAG-sirt1和 DR-GFP 质粒。利用特异性引物扩增出 SIRT6ΔC 和 δn 基因,克隆到质粒 pKH3HA (Addgene)和 pGex (GE 保健生命科学)载体中。通过将 SIRT6赖氨酸33转化为精氨酸(KR) ,或通过定点突变转化为谷氨酰胺(KQ)和 sirt6133组氨酸转化为酪氨酸(HY) ,获得了 SIRT6 KR、 KQ 和 HY 突变体。

Site-directed mutagenesis

定点突变

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The primers used for mutagenesis were designed using the online Quick Change Primer Design Program provided by Agilent Technologies. The mutagenesis was performed using Pfu DNA polymerase (Agilent) and 300 ng plasmid template, according to the manufacturer’s instructions. The PCR product was digested with DpnI endonuclease for 1 hr at 37°C, before transformation and sequencing.

用于诱变的引物是使用安捷伦科技有限公司提供的在线快速引物设计程序设计的。根据制造商的说明,用火球菌DNA聚合酶和300ng 质粒模板进行诱变。将 PCR 产物用核酸内切酶在37 ° c 下消化1小时,然后进行转化测序。

The following primers were used to generate the SIRT6 KR, KQ and HY mutants:

利用下列引物分别构建了 SIRT6 KR、 KQ 和 HY 突变体:

  • KR forward: 5’-ggagctggagcggagggtgtgggaact-3’前进: 5’-ggagctggggaggggggggggggggaact-3’
  • KR reverse: 5’-agttcccacaccctccgctccagctcc-3’KR 反向: 5’-agttcccccctgcccctcc-3’
  • KQ forward: 5′-ggagctggagcggcaggtgtgggaact-3’KQ 前锋: 5’-ggggggggggggggggggggaact-3’
  • KQ reverse: 5′-agttcccacacctgccgctccagctcc-3’KQ 反向: 5’-agttccacctctctctctctcctccc-3’
  • HY forward: 5′-acaaactggcagagctctacgggaacatgtttgtg-3’HY 前向: 5’-acaactcagctctacgggaatgtttg3’
  • HY reverse: 5′-cacaaacatgttcccgtagagctctgccagtttgt-3’5’-cacaacatgttccctgctgccgtgt-3’

Immunoprecipitation

免疫沉淀法

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HEK293T cells were transfected with the indicated plasmids using Lipofetamine3000 (Invitrogen, USA), according to the manufacturer’s instructions. The cells were lysed 48 hr post-transfection in lysis buffer [50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 0.2% NP40, 10% glycerol, 1 mM NaF, 1 mM Sodium butyrate, 10 mM Nicotinamide and a Complete protease inhibitor cocktail (Roche)]. The cell extracts were incubated with anti-FLAG M2 monoclonal antibody-conjugated agarose beads (Sigma) at 4°C overnight with rotation. The immunoprecipitates were boiled IN 2 × laemmli buffer and then analyzed by western blotting.

根据制造商的说明书,用 Lipofetamine3000(美国 Invitrogen)转染 HEK293T 细胞,获得上述质粒。细胞在转染后48小时在溶解缓冲液中溶解[50mm Tris-HCl,ph7.4,200mm NaCl,0.2% NP40,10% 甘油,1mm NaF,1mm 丁酸钠,10mm 烟酰胺和完整的蛋白酶抑制剂鸡尾酒(Roche)]。细胞提取物与抗 flag M2单克隆抗体共轭琼脂糖珠(Sigma)在4 ° c 条件下整夜旋转培养。免疫沉淀物煮沸后,用西方墨点法分析仪进行分析。

Chromatin immunoprecipitation (ChIP)

染色质免疫沉淀

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I-SceI-GR assays were performed as previously described (Soutoglou et al., 2007). HeLa cells stably transfected with DR-GFP were transiently transfected with RFP-I-SceI-GR together with FLAG-SIRT6, KR, KQ or HY. The cells were treated with 10−7 M triamcinolone acetonide (TA, Sangon, Shanghai) for 20 min, 48 hr after transfection, and then fixed with 1% paraformaldehyde at 37°C for 10 min to crosslink the chromatin. The reaction was stopped upon the addition of 0.125 M glycine. The chromatin was sonicated to 200 bps ~ 600 bps and incubated with the indicated antibodies. After de-cross linking, the ChIP-associated DNA was isolated and analyzed by quantitative real-time PCR (qRT-PCR).

I-SceI-GR 检测正如先前描述的(Soutoglou et al. ,2007)。将稳定转染 DR-GFP 的 HeLa 细胞与 FLAG-SIRT6、 KR、 KQ 或 HY 一起瞬时转染。细胞转染后用10-7m 曲安奈德(Sangon TA)处理20min,48hr,然后用1% 多聚甲醛固定于37 ° c 处交联10min。加入0.125 m 甘氨酸后,反应停止。染色质经超声波处理至200bps ~ 600bps,并与指示的抗体共同孵育。经去交联后,分离得到 chip 相关 DNA,并进行定量实时定量 PCR (qrt-PCR)分析。

Comet assay

彗星试验

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A comet assay was performed as previously described (Olive and Banáth, 2006). Briefly, after CPT treatment, the cells were digested into a single cell suspension, mixed with 1% agarose at the density of 1 × 105, coated on the slide and then incubated in lysis buffer (2% sarkosyl, 0.5M Na2EDTA, 0.5 mg/ml proteinase K) overnight at 37°C. The slides were incubated with N2 buffer (90 mM Tris, 90 mM boric acid and 2 mM Na2EDTA) and subjected to electrophoresis for 25 min at 0.6 V/cm. The slides were then incubated in staining solution containing 2.5 μg/ml propidium iodide for 30 min at room temperature. Images were captured under a fluorescent microscope.

彗星实验按照前面描述的方法进行(Olive 和 Banáth,2006)。简单地说,CPT 处理后,将细胞消化成单细胞悬液,加入1% 琼脂糖,密度为1 × 105,涂于载玻片上,在37 °c 的溶解液(2% sarkosyl,0.5 m Na2EDTA,0.5 mg/ml 蛋白酶 k)中过夜。采用 N2缓冲液(90mm Tris,90mm 硼酸和2mm Na2EDTA)对载玻片进行电泳,电泳时间为25min (0.6 V/cm)。将载玻片置于含2.5 μg/ml 碘化丙啶的染色液中室温孵育30分钟。图像是在荧光显微镜下拍摄的。

Cell fractionation

细胞分级

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Cells were scraped and washed with cold PBS. The cell pellet was resuspended in nuclei lysis buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.34M sucrose, 10% glycerol, 1 mM DTT, 0.1% TrionX-100.) for 10 min on ice and then centrifuged at 1300 g for 10 min. The pellet was resuspended in lysis buffer (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT) for 10 min on ice and centrifuged at 1700 g for 10 min. The pellet was saved as the chromatin fraction.

刮取细胞,用冷的 PBS 洗涤。细胞颗粒悬浮于细胞核裂解液中(10mm HEPES,10mm KCl,1.5 mM MgCl2,0.34 m 蔗糖,10% 甘油,1mm DTT,0.1% TrionX-100)在冰上放置10分钟,然后以1300克离心10分钟。用3mm EDTA、0.2 mM EGTA、1mm DTT 溶解液悬浮颗粒10min,1700g 离心10min。颗粒作为染色质部分保存下来。

MicroPoint laser irradiation and microscopy

微点激光照射与显微术

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U2OS cells or MEFs were seeded on a dish with a thin glass bottom (NEST), then locally irradiated with a 365 nm pulsed UV laser (16 Hz pulse, 56% laser output), generated by the MicroPoint Laser Illumination and Ablation System (Andor; power supply TPES24-T120MM, Laser NL100, 24V 50W), which is coupled to the fluorescence path of the Nikon A1 confocal imaging system (TuCam). Fluorescent protein recruitment and retention were continuously monitored by time-lapse imaging every 20 s for 10 min. The fluorescence intensity was quantified at each time-point using Fiji (Image J) software.

U2OS 细胞或 MEFs 被植入一个有薄玻璃底的培养皿(NEST) ,然后局部用365nm 脉冲紫外激光(16hz 脉冲,56% 激光输出)照射,该激光器由微点激光照明和烧蚀系统(Andor; 电源供应 TPES24-T120MM,激光 NL100,24V 50W)产生,与尼康 A1共聚焦成像系统(TuCam)的荧光路径耦合。荧光蛋白的补充和保留每20s 连续监测10min。用 Fiji (Image j)软件对每个时间点的荧光强度进行量化。

CRISPR/Cas9-mediated gene editing

Crispr/cas9介导的基因编辑

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CRISPR/Cas9-mediated gene editing was conducted as described (Ran et al., 2013). Briefly, a pX459 vector (Addgene#48139) was digested with BbsI and ligated with annealed oligonucleotides. The constructs containing the target gRNAs were transfected into HEK293T cells with Lipofetamine3000 (Invitrogen). The cells were selected for 5 days with puromycin 24 hr after transfection. Single clones were picked for sequencing.

Crispr/cas9介导的基因编辑是按照描述进行的(Ran 等人,2013)。用 BbsI 酶切 pX459载体(Addgene # 48139) ,并用退火寡核苷酸连接。将含有靶向 gRNAs 的构建体转染 HEK293T 细胞,用 Lipofetamine3000(Invitrogen)进行诱导。细胞转染后24小时用嘌呤霉素处理5天。选择单个克隆进行测序。

Peptide pulldown assay

肽下拉试验

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The C termini of H2AX (BGKKATQASQEY) and γH2AX (BGKKATQApSQEY) were synthesized and conjugated with biotin (GL Biochem, Shanghai). For one reaction, 1 μg biotinylated peptides was incubated with 1 μg GST-SIRT6 in binding buffer (50 mM Tirs-HCl, 200 mM NaCl, 0.05% NP40) overnight at 4°C. Streptavidin Sepharose beads (GE) was then used to pulldown the peptide and protein complexes for 1 hr at 4°C, and the samples were analyzed by western blotting.

合成了 H2AX (BGKKATQASQEY)和 γH2AX (BGKKATQApSQEY)的 c 端基,并与生物素(GL Biochem,上海)进行了共轭。在结合缓冲液(50mm Tirs-HCl,200mm NaCl,0.05% NP40)中,以1μg 的 GST-SIRT6为底物,在4 ° c 条件下连续孵育1μg 的生物素化多肽。链霉亲和素 Sepharose 珠(GE)用于将肽和蛋白复合物在4 ° c 下拉伸1小时,样品用西方墨点法分析仪分析。

Immunofluorescence staining

免疫荧光染色

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The cells were washed with PBS and fixed with 4% formaldehyde for 20 min, followed by permeabilization with cold methanol (−20°C) for 5 min and blocking with 5% BSA for 30 min. Then, the cells were incubated with primary antibodies (SIRT1, 1:200 dilution in 1% BSA; γH2AX, 1:500 dilution in 1% BSA; SIRT6, 1:200 dilution in 1% BSA) for 1 hr and secondary antibodies (donkey anti-rabbit IgG Alexa Fluor 594 and donkey anti-mouse IgG FITC from Invitrogen, 1:500 dilution in1% BSA) for 1 hr at room temperature in the dark. The cells were then co-stained with DAPI (Invitrogen) and observed under a fluorescent microscope.

4% 甲醛固定20min,冷甲醇(- 20 ° c)渗透5min,5% BSA 封闭30min。1% BSA,γh2ax,1:500稀释,1% BSA,SIRT6,1:200稀释,1% BSA 培养1hr,次级抗体(驴抗兔 IgG Fluor 594和驴抗小鼠 IgG FITC,1:500稀释,1% BSA 培养1hr。然后与 DAPI (Invitrogen)共染,在荧光显微镜下观察。

HR assay

心率测定

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U2OS cells stably transfected with DR-GFP were transfected with HA-I-SceI together with FLAG-SIRT6 WT, K33R, K33Q or H133Y. After transfection for 48 hr, the cells were harvested and the GFP-positive cell ratio per 104 cells was analyzed by flow cytometry (BD Biosciences). The relative HR efficiency was normalized to the vector control.

将 HA-I-SceI 与 FLAG-SIRT6 WT、 K33R、 K33Q、 H133Y 共同转染稳定转染的 U2OS 细胞。转染48小时后,采集细胞,流式细胞仪检测细胞中 gfp- 阳性细胞比例。将相对 HR 效率归一化为矢量控制。

Assessment of cell viability by MTS assay

MTS 法检测细胞活性

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Cell proliferation rate was examined using a CellTiter 96 AQueous Non-Radioactive Cell Proliferation assay (MTS) (Promega, USA). Approximately 2 × 103 cells/well were seeded in a 96-well plate and allowed to grow overnight. Cells were treated with CPT (0, 1 µM) for 1 hr or IR (2 Gy, 4 Gy, 6 Gy) and allowed to recover for 48 hr. MTS reagent (20 µL per well) was added, followed by incubation in the darkness at 37°C for 3 hr. The absorbance were measured at 490 nm using Bradford Reagent (Bio-Rad Laboratories). Cell viability was calculated as the ratio of absorbance of treated cells to control.

应用细胞滴度96水基非放射性细胞增殖试验(MTS)检测细胞增殖率。大约2 × 103/well 细胞接种在96孔板上,并在一夜之间生长。用 CPT (0,1μM)处理细胞1小时或 IR (2gy,4gy,6gy) ,恢复48小时。加入 MTS 试剂(每孔20μL) ,在37 °c 黑暗中培养3小时。用 Bradford 试剂(Bio-Rad 实验室)在490nm 处测量吸光度。细胞活力以处理细胞吸光度与对照细胞吸光度的比值计算。

Colony formation assay

集落形成试验

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HeLa cells were seeded into six-well plates 24 hr after transfection in defined numbers. Then, 24 hr after re-plating, the cells were exposed to increasing amounts of ionizing radiation delivered by an X-Rad 320 irradiator (Precision X-Ray Inc N. Branford, CT, USA). Fresh media was added after 7 days. Colonies containing at least 50 cells (10–14 days) were fixed with 20% methanol and stained with crystal violet and analyzed.

转染后24小时将 HeLa 细胞接种到6孔板中。然后,在重镀24小时后,电池暴露在由 X-Rad 320辐射器输送的越来越多的电离辐射中(美国 CT Branford 精密 x 射线公司)。7天后添加新鲜培养基。至少含有50个细胞(10-14天)的菌落用20% 甲醇固定,用结晶紫染色并进行分析。

DNA pulldown assay

脱氧核糖核酸下降试验

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The DNA binding assay was performed as previously described (Falck et al., 2005). Briefly, a biotin-conjugated DNA duplex 220 bp in size was generated by PCR amplification using biotin-labeled primers and a I-SceI plasmid as a template.

DNA 结合分析是进行了以前描述(Falck 等人,2005年)。采用生物素标记的引物和 I-SceI 质粒作为模板,通过 PCR 扩增得到一个大小为220bp 的生物素结合 DNA。

For the DNA pulldown assay, 10 pmol biotinylated DNA duplex was incubated with 0.5 μg of the indicated recombinant proteins in 300 μl binding buffer (10 mM Tris-Cl pH7.5, 100 mM NaCl, 0.01% NP40 and 10% glycerol) overnight at 4°C. Streptavidin Sepharose beads (GE) were added the next day, and incubated for another 1 hr with the samples. The beads were then collected and washed with binding buffer three times. The beads were subsequently boiled in 2 × laemmli buffer and analyzed by western blotting.

用10pmol 生物素化的重组蛋白在300μl 结合缓冲液(10mm Tris-Cl pH7.5,100mm NaCl,0.01% NP40和10% 甘油)中,在4 ° c 条件下连夜孵育10pmol 生物素化的 DNA。第二天加入链霉菌抗生素 Sepharose 珠子(GE) ,与样品一起再培养1小时。然后收集珠子,用装订缓冲液洗涤三次。这些珠子随后在2 × laemmli 缓冲液中煮沸,并由西方墨点法分析。

For linear and circular DNA competition assays, the ratios of the non-biotin labeled linear/circular DNA to the biotin DNA duplex were 5:1 or 10:1. Linear DNA was generated by PCR amplification using non-biotin-labeled primers, and circular DNA was obtained by cloning a PCR product into the pCDNA 3.1 plasmid (Invitrogen, USA).

在线性和环状 DNA 竞争实验中,非生物素标记的线性/环状 DNA 与生物素 DNA 的比值为5:1或10:1。采用非生物素标记引物进行 PCR 扩增,得到线性 DNA,并将 PCR 产物克隆到 pCDNA 3.1质粒(Invitrogen,USA)中,得到圆形 DNA。

The following sequences were used for PCR:

以下序列用于 PCR:

  • Forward, 5’-TACGGCAAGCTGACCCTGAA-3’前进,5’-tacggcaagctgctgaa-3’
  • Reverse, 5’-CGTCCTCCTTGAAGTCGATG-bio-3’反向,5’-cgtctctctcttgaagtcgatg-bio-3’

FP assay

荧光蛋白分析

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SIRT1, SIRT6 and SIRT7 recombinant proteins were purified in vitro, and incubated with a FAM-conjugated DNA duplex (20 nM) for 30 min on ice at the indicated concentration. The FP value of each sample was measured on 96 plates using a Multimode Plate Reader VictorTM X5 (PerkinElmer, USA) with an excitation wavelength of 480 nm and an emission wavelength of 535 nm. Curve fitting was performed in GraphPad prism.

SIRT1、 SIRT6和 SIRT7重组蛋白在体外纯化,并与一个 fam- 结合的双链 DNA (20nm)在指定浓度的冰上孵育30min。采用美国 PerkinElmer 公司的多模板读数器 VictorTM X5在96块平板上测量了各样品的荧光强度,激发波长为480nm,发射波长为535nm。曲线拟合采用石墨平板棱镜进行。

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