去乙酰化酶的协同作用直接识别 DNA 断裂并加强 DNA 损伤反应和修复

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A synergistic action of sirtuins directly recognizes DNA breaks and potentiates DNA damage response and repair

Summary

摘要

DNA damage response (DDR) is a highly orchestrated process; initially how the DNA breaks are recognized need in-depth study. Here, we show that polymerized SIRT6 deacetylase recognizes double-strand DNA breaks (DSBs) and potentiates DDR. SIRT1 deacetylates SIRT6 at residue K33, which is important for SIRT6 polymerization and mobilization toward DNA breaks. The K33-deacetylated SIRT6 anchors to γH2AX, allowing its retention on and subsequent remodeling of local chromatin. The K33R mutation, mimicking hypoacetylated SIRT6, rescues defective DNA repair imposed by SIRT1 deficiency in cells. Our data highlights a synergistic action of SIRTs in spatiotemporal regulation of DDR and DNA repair.

DNA 损伤反应(DDR)是一个高度协调的过程,最初如何识别 DNA 断裂需要深入研究。在这里,我们表明,高分子化的 SIRT6脱乙酰基酶识别双链 DNA 断裂(DSBs)并增强 DDR。SIRT1在 K33残基上脱乙酰化 SIRT6,这对 SIRT6的聚合和向 DNA 断裂方向的动员起重要作用。K33脱乙酰基化的 SIRT6锚定在 γh2ax 上,使其保留在局部染色质上并随后重塑。K33R 突变模仿低乙酰化的 SIRT6,对 SIRT1缺陷引起的细胞 DNA 修复缺陷具有保护作用。我们的数据突出了 SIRTs 在 DDR 和 DNA 修复的时空调节中的协同作用。

Introduction

引言

DNA damages are frequently induced by a variety of endogenous and exogenous agents. DNA damage response (DDR) is immediately elicited to ensure the genomic integrity, which is initiated by DNA break recognition, then followed by chromatin remodeling, signaling transduction and amplification (1). Double-strand breaks (DSBs) represent the most severe form of DNA lesions, 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, serving as a platform to orchestrate repair proteins to the vicinity of DNA breaks (2). Simultaneously, a variety of histone-modifying enzymes, heterochromatin factors and ATP-dependent chromatin remodelers cooperatively create a relaxed chromatin structure, allowing the access of additional repair factors to DSBs (3). Despite all the advances in understanding of DDR, how DSBs are initially and precisely recognized is largely unknown.

DNA 损伤常常是由多种内源性和外源性因素引起的。DNA 损伤反应(DNA damage response,DDR)是为了保证基因组的完整性而立即产生的,这种完整性是由 DNA 断裂识别引起的,然后是染色质重塑、信号转导和扩增。双链断裂(dsb)代表最严重的 DNA 损伤形式,被 Mre11-Rad50-Nbs1(MRN)复合体识别,该复合体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) ,作为一个平台协调修复 DNA 断裂附近的蛋白质(2)。同时,各种组蛋白修饰酶、异染色质因子和 atp 依赖性染色质重塑共同创建了一个松弛的染色质结构,允许其他修复因子进入 DSBs (3)。尽管在理解 DDR 方面取得了很多进展,但是 dsb 是如何被最初和准确地识别的在很大程度上还是未知的。

NAD+-dependent deacylase sirtuins regulate DDR, DNA repair and genomic integrity. Seven Sirtuins (SIRT1-7) with various enzymatic activities and physiological functions are identified in mammalian cells (4). Particularly, depletion of Sirt16 or 7 causes growth retardation, defective DDR and DNA repair and premature aging (57). Upon DNA damage, SIRT1 redistributes on chromatin, colocalized with γH2AX, and deacetylates XPA, NBS1 and Ku70, thus regulating nucleotide excision repair (NER), homologous recombination (HR) and non-homologous end-joining (NHEJ) (811). Depleting Sirt1 in mouse fibroblasts impairs DDR and leads to genomic instability (6). SIRT6 is one of the earliest factors recruited to DSBs, which initiates the subsequent recruitment of SNF2H, H2AX, DNA-PKcs and PARP1 (1215). SIRT6 mono-ribosylates PARP1 and thus to enhance its activity (16). Despite the rapid mobilization to DNA breaks, mechanisms initiating the recruitment of sirtuins are obscure (7,17,18).

NAD + 依赖的去酰化酶 sirtuins 调节 DDR、 DNA 修复和基因组完整性。在哺乳动物细胞中鉴定出7种具有多种酶活性和生理功能的 Sirtuins (SIRT1-7)(4)。特别是 Sirt1,6或7的缺失会导致生长迟缓,DDR 和 DNA 修复缺陷以及早衰(5-7)。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,从而调节核苷酸切除修复(NER)、同源重组(HR)和非同源末端连接(NHEJ)(8-11)。消耗小鼠成纤维细胞的 Sirt1基因,损害 DDR,导致基因组不稳定(6)。SIRT6是 dsb 最早招募的因子之一,启动了 SNF2H、 H2AX、 DNA-PKcs 和 PARP1(12-15)的后续招募。SIRT6单核糖基化 PARP1,从而增强其活性(16)。尽管快速动员 DNA 断裂,启动去乙酰化酶补充的机制是模糊的(7,17,18)。

Here, we found that SIRT6 polymerizes and directly recognizes DSBs via a putative DNA-binding pocket consisting of N- and C-termini from 2 adjacent molecules. SIRT1 interacts with SIRT6 and deacetylates it at K33, thus allowing its polymerization and recognition of DSBs. K33R mutant, mimicking hypoacetylated SIRT6, rescues DNA repair defects in SIRT1 KO cells. Our data highlight a synergistic action of Sirtuins in the spatiotemporal regulation of DDR.

在这里,我们发现 SIRT6通过一个由两个相邻分子的 n-和 C-termini 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 突变体对 SIRT1 KO 细胞 DNA 修复缺陷的保护作用。我们的数据突出了 Sirtuins 在 DDR 的时空调节中的协同作用。

Results

结果

SIRT6 directly recognizes DNA double-strand breaks

SIRT6能直接识别 DNA 双链断裂

Upon DNA damage, nuclear SIRTs (SIRT1/6/7) are quickly mobilized to DSBs (Figure S1), serving as a scaffold for DDR and DNA repair (7,17,18). Intriguingly, SIRTs are also activated by RNA and nucleosome (19,20). We reasoned that SIRTs might directly sense DNA breaks, especially DSBs. To test the hypothesis, a molecular docking simulation assay was performed using AutoDock Vina program (21). Crystal structures for SIRT1 (PDB code 4I5I) (22), SIRT6 (3PKI) (23) and SIRT7 (5IQZ) (24) were obtained from Protein Data Bank (https://www.rcsb.org). Heteroatoms were removed and Gasteiger charges were added to atoms. A DSB structure was extracted from PDB code 4DQY (25). As SIRTs use NAD+ as co-substrate in the amide bond hydrolysis, which shares similar skeleton of phosphate, base and ribose groups to the broken ends of DSB, we included NAD+ as simulation control. As shown (Figure 1A), the binding affinity between NAD+ and all nuclear SIRTs are within the range of −8 to −10 kcal/mol. Surprisingly, only the binding between DSB and SIRT6 is energetically favored (−12.7 kcal/mol), even lower than that of NAD+ (Figures 1A,B). This suggests a direct binding between DSB and SIRT6 and prompted us to gain further experimental evidences. We applied a DSB-mimicking biotin-conjugated DNA duplex and did in vitropulldown assay. Remarkably, recombinant SIRT6 (rSIRT6), but not rSIRT1 or rSIRT7, bounded to the DNA duplex (Figure 1C). This finding was verified by a fluorescence polarization (FP) assay using Fam-labeled DNA duplex. A dynamic fluorescence polarization was observed (Figure 1D, Kd = 166.3 nM), supporting a specific and direct binding of DNA duplex to rSIRT6. By contrast, fluorescence polarization was hardly detected for rSIRT1 or rSIRT7. To interrogate whether such binding is specific to broken DNA, the pulldown assay was again conducted in presence of unlabeled linear or circular DNA. While linearized DNA inhibited the binding of rSIRT6 to DNA duplex, circular one hardly did (Figure 1E). Together, the data indicate the direct recognition of DSB by SIRT6, but not SIRT1 or SIRT7.

DNA 损伤后,SIRT1/6/7可迅速被动员到 dsb 中(图 S1) ,作为 DDR 和 DNA 修复的支架(7,17,18)。有趣的是,sirt 也被 RNA 和核小体激活(19,20)。我们推断 sirt 可能直接感知 DNA 断裂,特别是 dsb。为了验证这一假设,使用 AutoDock Vina 程序(21)进行了分子对接模拟分析。从蛋白质数据库中获得了 SIRT1(PDB 代码4I5I)(22)、 SIRT6(3PKI)(23)和 SIRT7(5IQZ)(24)的晶体结构 https://www.rcsb.org。杂原子被去除,加斯泰格电荷被加到原子上。从 PDB 代码4DQY (25)中提取出 DSB 结构。以 NAD + 作为共底物进行酰胺键水解反应,使其与 DSB 的断端具有相似的磷酸基团、碱基团和核糖基团的骨架结构,并以 NAD + 作为模拟对照。如图1A 所示,NAD + 与所有核 sirt 之间的结合亲和力在 -8-10千卡/摩尔范围内。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1A,b)。这表明 DSB 和 SIRT6之间存在直接结合,并促使我们获得更多的实验证据。采用双链 DNA 模拟生物素结合双链 DNA,并进行了体外下拉实验。值得注意的是,重组的 SIRT6(rSIRT6) ,而不是 rSIRT1或 rSIRT7,与 DNA 双链结合(图1C)。这一发现被 fam 标记的 DNA 双链荧光偏振(FP)分析所证实。动态荧光偏振观察(图1D,Kd = 166.3 nM) ,支持特异性和直接结合的 DNA 复合体 rSIRT6。与此相反,rSIRT1和 rSIRT7的荧光偏振几乎没有检测到。为了确定这种结合是否是断裂 DNA 特有的,下拉实验再次在未标记的线性或圆形 DNA 存在下进行。线性化的 DNA 抑制 rSIRT6与 DNA 双链的结合,而环状 DNA 几乎没有抑制 rSIRT6与 DNA 双链的结合。结果表明,SIRT6能直接识别 DSB,而 SIRT1和 SIRT7不能直接识别 DSB。

Figure 1.

Figure 1. 图1SIRT6 directly recognizes DNA breaks. SIRT6可以直接识别 DNA 碎片

(A) Predicted binding affinity (kcal/mol) between sirtuins (SIRTs) and ligands.

(a)预测 sirtuins (SIRTs)与配体的结合亲和力(kcal/mol)。

(B) Molecular docking of DSB (right) and NAD+ (left) with SIRT6.

(b) DSB (右)和 NAD + (左)与 SIRT6的分子对接。

(C) Biotin-labeled DNA duplex were incubated with indicated recombinant SIRTs. Streptavidin beads pulldown was blotted with anti GST and anti SIRT1 antibodies.

(c)生物素标记的双链 DNA 与特异性重组 SIRTs 共同孵育。用抗 GST 和抗 SIRT1抗体对链霉亲和素珠子进行下拉实验。

(D) Fluorescence polarization (FP) of Fam labeled DNA was detected after incubating with GST-SIRT1, GST-SIRT6 or GST-SIRT7.

(d)与 GST-SIRT1、 GST-SIRT6或 GST-SIRT7孵育后,荧光偏振(FP)检测家族标记 DNA。

(E) Pulldown assay of Biotin-labeled DNA duplex with GST-SIRT6 in the presence of unlabeled linear DNA or circular DNA.

(e)用 GST-SIRT6对生物素标记的双链 DNA 在非标记线性 DNA 和圆形 DNA 存在下的下拉实验。

Dynamic K33 (de)acetylation regulates SIRT6-sensing DSBs

动态 K33(de)乙酰化调节 sirt6感受 DSBs

As predicted from the crystallographic data, SIRT6 forms asymmetric hexamer (23), generating three potential DSB binding pockets; each consists of two N-termini and two C-termini from two adjacent molecules (Figure S2A). Both N- and C-termini are essential for the chromatin association of SIRT6 (26). To gain biochemical evidence of SIRT6 polymerization, we employed a biomolecule fluorescence compensation system (BiFC). SIRT6 cDNA was cloned to either the N-terminal or C-terminal of a yellow fluorescence protein (YFP), namely N-SIRT6 and C-SIRT6. Yellow fluorescence is detectable by FACS only when N-SIRT6 directly interacts with C-SIRT6. These constructs were co-transfected into HEK293 cells. As shown, a strong fluorescence signal was detected by FACS in more than 24% cells (Figure S2B), suggesting a direct interaction between SIRT6 molecules. Such polymerization of SIRT6 was confirmed by co-immunoprecipitation (Co-IP) in HEK293 cells wherein FLAG-SIRT6 and HA-SIRT6 were co-overexpressed. As shown, FLAG-SIRT6 was detected in the anti HA-SIRT6 immunoprecipitates (Figure S2C).

正如晶体学数据所预测的那样,SIRT6形成了不对称的六晶体(23) ,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 S2A)。N-和 c- 末端蛋白对于 SIRT6(26)的染色质联合是必不可少的。为了获得 SIRT6聚合反应的生化证据,我们采用了生物分子荧光补偿系统(BiFC)。将 SIRT6基因克隆到一个黄色荧光蛋白(YFP)的 n 端或 c 端,即 n-SIRT6和 c-SIRT6。只有当 N-SIRT6与 C-SIRT6直接相互作用时,FACS 才能检测到黄色荧光。这些构建物共转染 HEK293细胞。如图所示,流式细胞仪在24% 以上的细胞中检测到强荧光信号(图 S2B) ,提示 SIRT6分子之间存在直接相互作用。免疫共沉淀(Co-IP)证实了 SIRT6在 HEK293细胞中的聚合,其中 flag-SIRT6和 ha-SIRT6共表达。如图所示,在抗 HA-SIRT6免疫沉淀物中检测到了 FLAG-SIRT6(图 S2C)。

The phosphate backbone of DSB is negative charged. A positive-charged environment in SIRT6 favors its binding to DSBs. Indeed, one predicted DSB-binding pocket formed by two adjacent molecules in SIRT6 hexamer consists of six positive-charged residues at the edge, i.e. 4 arginines (R32/39) and 2 lysines (K33) (Figure S2D). Acetylation belongs to the most redundant posttranslational modifications, which can turn positive-charged K to neutral Kac. This property is utilized by proteins with lysine-rich domain (KRD), e.g. Histones, Ku70 and p53, for dynamic interaction with proteins harboring acidic domain like SET (27). The heterodimerized Ku70 and Ku80 directly senses DSBs by the flexible C-termini with multiple Ks, and regulates non-homologous end joining (NHEJ) (28). We therefore examined whether SIRT6 is (de)acetylated. FLAG-SIRT6 was immunoprecipitated with anti-FLAG antibody and blotted with anti Kac antibodies. As shown, acetylated Ks was detected in the precipitated FLAG-SIRT6 (Figure 2A). We further purified FLAG-SIRT6 and did high-resolution LC-MS/MS to identify Ks undergoing acetylation. Potential acetylated Ks were summarized (Table S1). In N-terminus, K15 and K33 were identified. To confirm these acetylated Ks, we did point mutagenesis on K15/17/33; K17 was included as control. While neither K15R nor K17R affected the acetylation level of FLAG-SIRT6, K33R significantly inhibited it (Figures 2A and S3), supporting that K33 undergoes dynamic (de)acetylation.

DSB 的磷酸盐主链带负电荷。SIRT6中的正电荷环境有利于它与 DSBs 的绑定。事实上,有人预测 SIRT6六聚体中两个相邻分子形成的 dsb 结合囊包含6个边缘带正电荷的残基,即4个精氨酸(R32/39)和2个赖氨酸(K33)(图 S2D)。乙酰化反应属于翻译后修饰中最冗余的修饰,它能将正电荷的 k 转化为中性的 Kac。这一特性被富含赖氨酸结构域(KRD)的蛋白质利用,例如组蛋白、 Ku70和 p53,用于与包含酸性结构域(SET (27))的蛋白质进行动态相互作用。异质二聚体 Ku70和 Ku80通过具有多个 Ks 的柔性 c 端口直接感应 dsb,并调节非同源性末端接合(NHEJ)(28)。因此,我们检测了 SIRT6是否(去)乙酰化。用抗 flag 抗体免疫促进 FLAG-SIRT6的表达,并用抗 Kac 抗体进行印迹。如图所示,在沉淀的 FLAG-SIRT6中检测到乙酰化的 Ks (图2A)。我们进一步纯化了 FLAG-SIRT6,并进行了高分辨液相色谱-串联质谱(LC-MS/MS)的鉴定。总结了潜在的乙酰化酶(表 S1)。在 n 端,鉴定出 K15和 K33。以 K15/17/33为材料,以 K17为对照,对这些乙酰化的 Ks 进行点突变。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 S3) ,支持 K33发生动态乙酰化。

Figure 2.

Figure 2. 图2SIRT6 K33 (de)acetylation regulates the DSB binding SIRT6 K33(de)乙酰化调节 DSB 结合

(A) The acetylation of WT and K33R mutated SIRT6.

(a)野生型和 K33R 突变型 SIRT6的乙酰化。

(B) Pulldown assay of Biotin-labeled DNA duplex with indicated GST-SIRT6.

(b) GST-SIRT6对生物素标记的双链 DNA 的下拉实验。

(C) Fluorescence polarization (FP) of Fam labeled DNA was detected after incubating with GST-SIRT6 K133R or K133Q.

(c)与 GST-SIRT6 K133R 或 K133Q 共孵育后荧光偏振(FP)检测。

(D-E) Dynamic recruitment of GFP-SIRT6, K33R, K33Q and HY (H133Y) to the laser-induced DNA breaks. Representative images were shown (D) and the white dot circles indicate the damage sites. The relative intensity was calculated by software Fiji (Image J®(E).

(D-E) GFP-SIRT6、 K33R、 K33Q 和 HY (H133Y)对激光诱导 DNA 断裂的动态补充。有代表性的图像显示(d) ,白点圆圈表示损害部位。相对强度由 Fiji 软件计算(图 j)(e)。

(F) Schematic map of DR-GFP construct, which contains a single I-SceI site to create DNA break in the presence of triamcinolone acetonide and I-SceI endonuclease. The amplification primers 2K and 5K downstream I-SceI site used for q-PCR were labeled.

(f) DR-GFP 结构示意图,其中包含一个 I-SceI 位点,在曲安奈德和 I-SceI 核酸内切酶存在的情况下产生 DNA 断裂。对用于 q-PCR 的扩增引物2K 和5K 下游 I-SceI 位点进行了标记。

(G) Successful generation of DNA breaks in DR-GFP stably transfected Hela cells after triamcinolone acetonide (TA) treatment for 20min, evidenced by elevated γH2AX staining.

(g)用曲安奈德(TA)处理 Hela 细胞20min 后,在稳定转染的 DR-GFP 细胞中成功地产生 DNA 断裂,可见 γh2ax 染色升高。

(H) ChIP-PCR analysis showing the enrichment of SIRT6 and various mutants at the vicinity of DNA breaks. Relative expression of SIRT6 were confirmed by Western blotting. Q-PCR data was normalized to Input DNA and sample without treatment of I-SceI endonuclease (no cut). *P < 0.05.

(h) ChIP-PCR 分析表明,SIRT6和各种突变体在 DNA 断裂附近均有富集。目的: 探讨 SIRT6基因在大肠杆菌中的表达及其意义。Q-PCR 数据在未经酶切处理的情况下归一化为输入 DNA 和标本。* p < 0.05.

(I) Cell fraction analysis showing chromatin enrichment of SNF2H, SIRT6 in FLAG-SIRT6, K33R, K33Q and HY reconstituted SIRT6 KO HEK293T cells.

(i)细胞分数分析表明,在 flag-SIRT6、 K33R、 K33Q 和 HY 重组的 SIRT6 KO HEK293T 细胞中,SNF2H、 SIRT6染色质富集。

To understand the function of K33 acetylation, we examined whether it is required for the DSB binding of SIRT6. We mutated lysine to arginine (K33R) or glutamine (K33Q) to mimic the deacetylated or acetylated SIRT6 (29). SIRT6 histidine 133 was mutated to tyrosine (H133Y, HY) to blunt SIRT6 enzyme activity (18). The binding of K33Q and H133Y to the DNA duplex was significantly compromised compared to WT and K33R (Figure 2B). Consistently, fluorescence polarization was recorded for SIRT6 K33R (Kd = 104.9 nM), which was hardly detected in case of SIRT6 K33Q (Figure 2C). Of note, H133 is critical for chromatin enrichment of SIRT6 (26). We further monitored GFP-SIRT6 mobility upon DNA damage. GPF-SIRT6 WT, K33Q, K33R and H133Y were reconstituted in Sirt6−/− cells and their recruitment to DSBs was monitored. While K33Q and H133Y significantly jeopardized the efficient recruitment to DNA breaks, K33R completely retained such ability (Figures 2D-E). To gain more experimental support, we applied an inducible DR-GFP reporter system, which 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, evidenced by increased γH2AX level (Figure 2F-G). The occupancy of SIRT6 on chromatin surrounding DSBs is detected by chromatin immunoprecipitation (ChIP) and quantitative PCR (30). The result showed that both K33Q and H133Y compromised the recruitment of SIRT6 to the sites of damage, whereas K33R remained as efficient as WT (Figures 2H). Of note, K33Q and H133Y also differed the recruitment of SNF2H to DSBs (Figure 2I), which is accomplished by SIRT6 (18). By contrast, SNF2H recruitment was merely altered by K33R. Neither K33R nor K33Q affected the deacetylase activity of SIRT6 (Figure S4).

为了解 K33的乙酰化作用,我们研究了 SIRT6的 DSB 结合是否需要 K33的乙酰化作用。我们将赖氨酸突变为精氨酸(K33R)或谷氨酰胺(K33Q) ,以模拟去乙酰化或乙酰化的 SIRT6(29)。SIRT6组氨酸133突变为酪氨酸(H133Y,HY) ,使 SIRT6酶活性减弱(18)。与 WT 和 K33R 相比,K33Q 和 H133Y 与 DNA 双链的结合明显受损(图2B)。SIRT6 K33R (Kd = 104.9 nM)的荧光偏振度一致,而 SIRT6 K33Q (图2C)的荧光偏振度很低。值得注意的是,H133是至关重要的染色质丰富的 SIRT6(26)。我们进一步监测 GFP-SIRT6移动性对 DNA 损伤。在 Sirt6-/-细胞中重组 GPF-SIRT6 WT、 K33Q、 K33R 和 H133Y,并监测它们对 dsb 的补充。虽然 K33Q 和 H133Y 显著地损害了有效补充 DNA 断裂,K33R 完全保留这种能力(图2D-E)。为了获得更多的实验支持,我们应用了一个可诱导的 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,其表现为 γh2ax 水平升高(图2F-G)。SIRT6在 DSBs 周围染色质上的占位通过染色质免疫沉淀和定量 PCR (30)检测。结果表明,K33Q 和 H133Y 均能抑制 SIRT6在损伤部位的补充,而 K33R 与 WT 一样有效(图2H)。值得注意的是,K33Q 和 H133Y 也不同于 SNF2H 对 dsb 的补充(图2I) ,这是由 SIRT6(18)完成的。相比之下,SNF2H 的补充只是被 K33R 改变。K33R 和 K33Q 均不影响 SIRT6的脱乙酰酶活性(图 S4)。

We next analyzed whether dynamic K33 (de)acetylation modulates the polymerization of SIRT6. HA-SIRT6 and various FLAG-SIRT6 mutants were co-overexpressed and Co-IP was performed. FLAG-SIRT6 was observed in the anti-HA immunoprecipitates, supporting polymerization of SIRT6 (Figure S2C). While HA-SIRT6 bonded to K33R to a similar extent as WT, its binding to K33Q was significantly jeopardized. Of note, H133Y, the enzyme-dead mutation, also jeopardized polymerization of SIRT6. This is indeed consistent with the finding that H133, in addition to the deacetylase activity of SIRT6, is important for its chromatin association (26). The jeopardized polymerization is confirmed by BiFC assay (Figure S5A,B). Together, the data implicate that dynamic 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的聚合反应。对 HA-SIRT6和各种 FLAG-SIRT6突变体进行了共表达,并进行了 Co-IP 检测。在抗 ha 的免疫沉淀物中观察到 FLAG-SIRT6,支持 SIRT6的聚合(图 S2C)。HA-SIRT6与 K33R 的结合程度与 WT 相似,但其与 K33Q 的结合受到严重损害。值得注意的是,H133Y 这种酶死突变也危害到 SIRT6的聚合。这与 H133,除了 SIRT6的去乙酰化酶活性之外,对于其染色质结合也很重要的发现是一致的(26)。聚合的危害性由 BiFC 分析确定(图 S5A,b)。总之,这些数据表明动态 K33(去)乙酰化可以调节 SIRT6的聚合,从而与 DSB 结合。

SIRT6 is a deacetylation target of SIRT1

SIRT6是 SIRT1的去乙酰化靶点

We noticed that the level of acetyl SIRT6 was largely elevated in the presence of class III HADC (SIRTs) inhibitor nicotinamide (NAM) or SIRT1-specific inhibitor Ex527, but not class I/II HADC inhibitor Trichostatin A (TSA) (Figure S6). This suggests that SIRT1 is likely involved in SIRT6 deacetylation. Indeed, Co-IP and Western blotting revealed that FLAG-SIRT6 interacted with endogenous SIRT1 (Figure 3A) and FLAG-SIRT1 interacted with endogenous SIRT6 in HEK293 cells (Figure 3B). In addition, SIRT1 was detected in the anti SIRT6 immunoprecipitates and vice versa (Figure 3C,D). Determined by GST pulldown assay, His-SIRT1 was pull down by GST-SIRT6 in the test tubes (Figure 3E). Further, co-localization of SIRT6 and SIRT1 was evidenced by confocal microscopy in cells co-transfected with GFP-SIRT6 and DsRed-SIRT1 or co-stained with specific antibodies (Figures 3F and S7A).

我们注意到,在含有 III 类 HADC 抑制剂(SIRTs)烟酰胺(NAM)或 sirt1特异性抑制剂 Ex527的情况下,血清乙酰化 SIRT6水平明显升高,而不含 I/II 类 HADC 抑制剂曲古菌素 a (TSA)(图 S6)。这表明 SIRT1可能参与了 SIRT6的脱乙酰化。实际上,Co-IP 和西方墨点法显示,在 HEK293细胞中,FLAG-SIRT6与内源性 SIRT1(图3 a)和 flag-SIRT1相互作用,与内源性 SIRT6相互作用(图3 b)。此外,在抗 SIRT6的免疫沉淀物中检测到 SIRT1,反之亦然(图3 c,d)。用 GST 下拉法测定,在试管中用 GST-sirt6将 His-SIRT1下拉(图3E)。此外,SIRT6和 SIRT1在 gfp-SIRT6和 dsred-SIRT1共转染或特异性抗体共染的细胞中的共定位被证实为共聚焦显微镜。

Figure 3.

Figure 3. 图3SIRT6 interacts with SIRT1. SIRT6与 SIRT1相互作用

(A) Western blots showing SIRT1 in anti-FLAG immunoprecipitates in HEK293 cells transfected with FLAG-SIRT6 or empty vector.

(a)用 FLAG-SIRT6或空载体转染 HEK293细胞,在抗 flag 免疫沉淀物中检测到 SIRT1蛋白。

(B) Western blots showing SIRT6 in anti-FLAG immunoprecipitates in HEK293 cells transfected with FLAG-SIRT1 or empty vector.

(b)用 FLAG-SIRT1或空载体转染 HEK293细胞,可见 SIRT6蛋白在抗 flag 免疫沉淀物中的表达。

(C) Western blots showing SIRT1 in anti-SIRT6 immunoprecipitates in Hela cells. (D) Western blots showing SIRT6 in anti-SIRT1 immunoprecipitates in Hela cells.

(c)免疫印迹显示 SIRT1在 Hela 细胞抗 sirt6免疫沉淀物中的表达。(d)免疫印迹显示 SIRT6在 Hela 细胞抗 sirt1免疫沉淀物中的表达。

(E) GST pulldown assay showing the interaction between GST-SIRT6 and His-SIRT1 in vitro.

(e) GST-sirt6与 His-SIRT1体外相互作用的 GST 下拉实验。

(F) Representative images showing colocalized DsRed-SIRT1 and GFP-SIRT6 in U2OS cells, determined by confocal microscopy. Scale bar, 10 μm.

(f)代表性图像显示 U2OS 细胞中共定位的 red-sirt1和 GFP-SIRT6,共聚焦显微镜: 10μm。

(G) Co-immunoprecipitation and Western blotting data showing interaction between FLAG-SIRT1 and HA-SIRT6 ΔN (N-terminus deleted), ΔC (C-terminus deleted) and ΔCN (N-/C-termini deleted) in HEK293 cells.

(g)在 HEK293细胞中,共免疫沉淀和西方墨点法数据显示 FLAG-SIRT1与 HA-SIRT6 δn (n 端缺失)、 δc (c 端缺失)和 δcn (n-/c 端缺失)之间的相互作用。

SIRTs contain a conserved Sir2 domain and flexible N- and C-termini. To locate exact SIRT6 domains that interact with SIRT1, we did domain mapping by serially mutating the N- and C-terminus as reported (26) (Figure S7B,C). Western blotting analysis showed that the interaction between SIRT6 and SIRT1 was completely abolished in case that N- or C-terminus-deleted SIRT6 was examined (Figure 3G). The data indicate that SIRT6 physically interacts with SIRT1.

SIRTs 包含一个保守的 Sir2结构域和灵活的 n-和 c- 末端。为了准确定位与 SIRT1相互作用的 SIRT6域,我们通过连续变异 n 端和 c 端来进行域映射(见图 S7B,c)。西方墨点法分析显示,如果检查 n 或 c 末端缺失的 SIRT6,SIRT6和 SIRT1之间的相互作用就完全消失了(图3 g)。数据表明,SIRT6相互作用(分子生物学) SIRT1。

We next examined whether SIRT1 deacetylates SIRT6. As shown, the overexpression of SIRT1 but not other sirtuins inhibited the acetylation of FLAG-SIRT6 (Figure 4A). On the other front, knocking down SIRT1 significantly upregulated the acetylation level of endogenous SIRT6 in HEK293 cells (Figure 4B). Further, the acetylation level of SIRT6 was decreased in the presence of ectopic SIRT1 rather than its catalytic mutant SIRT1-H366Y (Figure 4C), suggesting SIRT6 likely as a deacetylation target of SIRT1. To test it directly, an in vitro deacetylation assay was employed. Recombinant FLAG-SIRT6 was eluted with FLAG peptide from HEK293 cell lysate. SIRT1 deacetylated SIRT6 in the presence of NAD+, while NAM inhibited this process (Figure 4D). The deacetylase-inactive SIRT1-HY(S355A) was unable to deacetylate SIRT6. As SIRT1 interacts with the N-terminus of SIRT6, it might deacetylate K33ac. While the acetylation level of SIRT6 was increased in SIRT1−/− cells, that of K33R was hardly affected (Figure 4E). Additionally, the acetylation level of SIRT6 K33R was rarely changed upon SIRT1 overexpression (Figure 4F), whereas that of K143/145R was downregulated by ectopic SIRT1 (Figure S8A), supporting K33ac as the main target of SIRT1. By contrast, SIRT1 acetylation level was merely affected when overexpressing SIRT6 in cells (Figure S8B). To further validate the findings, we synthesized a K33ac-containing peptide, and found that it effectively blocked the in vitro binding of SIRT6 to SIRT1 (Figure 4G). Of note, GST pulldown assay suggests that the N-terminus rather than the C-terminus of SIRT6 is responsible for its interaction with SIRT1. Together, the data suggest that SIRT1 deacetylates SIRT6 at K33.

我们接下来检测 SIRT1是否脱乙酰基。结果表明,SIRT1的过表达抑制了 FLAG-SIRT6的乙酰化(图4A)。另一方面,敲除 SIRT1可显著提高 HEK293细胞内源性 SIRT6的乙酰化水平(图4B)。此外,SIRT6的乙酰化水平在异位 SIRT1的存在下降,而不是其催化突变体 SIRT1-h366y (图4C) ,提示 SIRT6可能是 SIRT1的脱乙酰化靶点。采用体外脱乙酰度法直接测定。用 HEK293细胞裂解液中的 FLAG 多肽洗脱重组 FLAG-SIRT6。SIRT1在 NAD + 存在下脱乙酰化 SIRT6,而 NAM 抑制了这一过程(图4D)。去乙酰化酶非活性的 SIRT1-HY (S355A)不能脱乙酰化 SIRT6。由于 SIRT1与 SIRT6的 n 端相互作用,可能是脱乙酰基的 K33ac。SIRT1-/-细胞中 SIRT6的乙酰化水平升高,而 K33R 的乙酰化水平几乎没有受到影响(图4E)。此外,SIRT1过表达后,SIRT6 K33R 的乙酰化水平很少发生改变(图4F) ,而 K143/145R 的乙酰化水平则被异位的 SIRT1下调(图 S8A) ,以 K33ac 为 SIRT1的主要靶点。相比之下,细胞中过量表达 SIRT6仅影响 SIRT1乙酰化水平(图 S8B)。为了进一步验证这些发现,我们合成了一个含有 k33ac 的多肽,发现它能有效地阻断 SIRT6与 SIRT1的体外结合(图4G)。值得注意的是,GST 下拉实验表明,SIRT6的 n 端而不是 c 端负责与 SIRT1的相互作用。总之,数据表明,SIRT1脱乙酰化 SIRT6在 K33。

Figure 4.

Figure 4. 图4SIRT1 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.

(a)外源性表达 SIRT1-5和 SIRT7的 HEK293细胞中 FLAG-SIRT6的乙酰化水平。

(B) The acetylation level of endogenous SIRT6 in HEK293 cells treated si-SIRT1 or scramble (Sram) siRNAs.

(b)经 si-SIRT1或 scramble (Sram) siRNAs 处理的 HEK293细胞内源性 SIRT6的乙酰化水平。

(C) The acetylation level of FLAG-SIRT6 in SIRT1−/− cells reconstituted with SIRT1 or enzyme-inactive H363Y.

(c) SIRT1和 H363Y 重组细胞中 FLAG-SIRT6的乙酰化水平。

(D) The acetylation level of FLAG-SIRT6 in presence of SIRT1, H355A, NAD+ (500 μM) and/or NAM (2 mM).

(d)在 SIRT1、 H355A、 NAD + (500μM)和/或 NAM (2mm)存在下,FLAG-SIRT6的乙酰化水平。

(E) The acetylation level of FLAG-SIRT6 and K33R in SIRT1−/− and WT HEK293 cells.

(e) SIRT1-/-和 WT HEK293细胞中 FLAG-SIRT6和 K33R 的乙酰化水平。

(F) The acetylation level of FLAG-SIRT6 and K33R in HEK293 cells with or without ectopic SIRT1.

(f)有无异位 SIRT1时 HEK293细胞中 FLAG-SIRT6和 K33R 的乙酰化水平。

(G) GST pulldown assay with GST-SIRT6 WT, ΔN, ΔC and His-SIRT1 in presence or absence of 10 μM K33ac peptide (PEELERK(ac)VWELARL), which represents a 14-aa peptide containing acetylated K33 of SIRT6.

(g)用 GST-SIRT6 WT、 δn、 δc 和 His-SIRT1检测10μM K33ac 多肽(PEELERK (ac) VWELARL)的 GST 下拉作用。

γH2AX ensures SIRT6 retention on DSBs

γh2ax 确保 SIRT6在 DSBs 上的保留

Since the enrichment of SIRT6 at DNA breaks, we asked whether γH2AX was involved in this event. γH2AX is dispensable for the initial DSB recognition but serves as a platform for recruiting DDR factors (2). We thus did Co-IP of endogenous SIRT6 in cells treated with or without CPT. Interestingly, H2AX and γH2AX were detected in anti SIRT6 precipitates only when cells were treated with CPT (Figure 5A,B). Further, in vitro pulldown assay with biotin-labeled a C-terminal peptide of γH2AX and H2AX was performed. Consistently, GST-SIRT6 recognized the peptide of γH2AX instead of H2AX (Figure 5C). To study the interacting domain, we purified truncated GST-SIRT6. Peptide pulldown assay revealed that N-terminus truncation was enough to abolish the binding of SIRT6 to γH2AX peptide, while the deletion of C-terminus had little effect (Figure 5D). We then investigated whether SIRT1-mediated deacetylation contributes to the binding of SIRT6 to γH2AX. As shown, K33R mutant efficiently bound to γH2AX in similar extent to WT, but that was abolished in case of K33Q (Figure 5E).

由于 SIRT6在 DNA 断裂时富集,我们询问 γh2ax 是否参与了这一事件。γh2ax 对于最初的 DSB 识别是可有可无的,但它同时也是一个招募 DDR 因子的平台(2)。因此,我们对内源性 SIRT6进行了经 CPT 处理和不经 CPT 处理的细胞的 Co-IP 检测。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)。此外,用生物素标记的 γH2AX 和 H2AX 的 c 末端肽段进行体外下拉实验。GST-SIRT6识别 γH2AX 多肽而不是 H2AX (图5C)。为了研究相互作用结构域,我们纯化了截短型 GST-SIRT6。肽段下拉实验表明,n 端截断足以阻断 SIRT6与 γh2ax 肽的结合,而 c 端缺失则无明显影响(图5D)。然后我们研究了 sirt1介导的去乙酰化是否参与了 SIRT6与 γh2ax 的结合。结果表明,K33R 突变体与 γh2ax 的结合强度与 WT 相似,但 K33Q 突变体与 γh2ax 的结合强度较弱(图5E)。

Figure 5.

Figure 5. 图5γH2AX is required for the chromatin retention of SIRT6. SIRT6的染色质保留需要 γh2ax 参与

(A,B) Representative immunoblots showing H2AX (A) and γ-H2AX (B) in the anti-SIRT6 immunoprecipitates in HEK293 cells treated with or without camptothecin (CPT).

(a,b) HEK293细胞经喜树碱(CPT)处理后,表现为 H2AX (a)和 γ-H2AX (b)的代表性免疫印迹。

(C) Pulldown assay and Western blotting data showing interaction between GST-SIRT6 and synthesized γH2AX but not H2AX peptide.

(c)下拉实验和西方墨点法数据显示 GST-SIRT6与人工合成的 γH2AX 之间的相互作用,但不显示 H2AX 肽。

(D) Pulldown assay and Western blotting showing interaction between synthesized γH2AX peptide and GST-SIRT6 WT and truncated form (ΔN and ΔC).

(d)下拉实验和西方墨点法显示合成的 γh2ax 多肽与 GST-SIRT6 WT 和截短型(δn 和 δc)之间的相互作用。

(E) Pulldown assay and Western blotting showing interaction between γH2AX peptide and GST-SIRT6 WT, K33R and K33Q.

(e)下拉实验和西方墨点法显示 γh2ax 肽与 GST-SIRT6 WT、 K33R 和 K33Q 之间的相互作用。

(F,G) Laser micropointer analysis of SIRT6 recruitment in H2AX+/+ and H2AX−/− MEFs

(f,g)激光微指针分析 H2AX +/+ 和 H2AX-/-MEFs 中 SIRT6的补充

(F), and in H2AX−/− MEFs reconstituted with H2AX WT, S139D mimicking hyper-phosphorylation or S139A mimicking hypo-phosphorylation (G). PAR immunostaining reveals the damage site. Scale bar, 10 μm.

(f) ,在 H2AX 重组的 H2AX-/-MEFs 中,S139D 模拟过磷酸化或 S139A 模拟过磷酸化(g)。PAR 免疫染色显示损伤部位。比例尺,10微米。

To investigate the functional relevance of SIRT6-γH2AX interaction, we applied laser-induced DNA damage assay and tracked re-location of SIRT6 in MEFs lacking H2AX by immunofluorescence microscopy. As shown, GFP-SIRT6 was immediately recruited to DNA lesions in H2AX+/+ and H2AX−/− MEFs (Figure 5F), implicating that H2AX is dispensable for the initial recruitment of SIRT6. Interestingly, GFP-SIRT6 diminished 10 min after the laser treatment in H2AX−/− MEFs but persisted in H2AX+/+ cells. H2AX is rapidly phosphorylated at serine 139 in response to DSBs (31). When H2AX WT, S139A and S139D were re-introduced into H2AX−/− MEFs, the retention of SIRT6 was restored in WT and S139D-re-expressing cells but not S139A (Figure 5G). Together, these data indicate that SIRT6 recognizes γH2AX surrounding DSBs, which is enhanced by SIRT1-mediated deacetylation.

为了研究 SIRT6-γH2AX 相互作用的功能相关性,我们应用激光诱导 DNA 损伤实验,通过免疫荧光显微镜追踪 SIRT6在缺乏 H2AX 的 MEFs 中的重新定位。如图所示,GFP-SIRT6被立即招募到 H2AX +/+ 和 H2AX-/-MEFs (图5F)的 DNA 损伤中,这表明 H2AX 对于 SIRT6的初始招募是可有可无的。有趣的是,GFP-SIRT6在激光照射 H2AX-/-MEFs 10分钟后减少,但在 H2AX +/+ 细胞中仍然存在。针对 DSBs (31) ,H2AX 在丝氨酸139处迅速磷酸化。当 H2AX WT、 S139A 和 S139D 重新引入 H2AX-/-MEFs 时,SIRT6在 WT 和 S139D–re 表达细胞中的保留得到恢复,而 S139A 未恢复(图5G)。总之,这些数据表明,SIRT6识别周围的 γh2ax DSBs,这是由 sirt1介导的脱乙酰化增强。

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 investigate whether SIRT1 and SIRT6 cooperatively modulate DDR and DNA repair. The DR-GFP reporter system and ChIP-PCR analysis were applied. The recruitment of FLAG-SIRT6 to DSB vicinity was significantly reduced when SIRT1 was silenced by siRNA (Figures 6A-B). We further analyzed the dynamic recruitment of GFP-SIRT6 upon laser-induced DNA damage. GFP-SIRT6 was rapidly recruited to DNA breaks in WT cells, but this process was largely deferred in Sirt1−/− cells (Figures 6E-F), suggesting an indispensable role of SIRT1 in the initial recruitment of SIRT6 to DSBs. By contrast, the recruitment of SIRT1 to DSBs was merely affected by SIRT6 downregulation, as determined by ChIP-PCR analysis and laser micropointer assay (Figures 6C-D,G).

SIRT1和 SIRT6之间的物理相互作用促使我们研究 SIRT1和 SIRT6是否协同调节 DDR 和 DNA 修复。应用 DR-GFP 报告系统和 ChIP-PCR 分析。当 siRNA 沉默 SIRT1时,FLAG-SIRT6在 DSB 附近的补充量显著减少(图6A-B)。我们进一步分析了 GFP-SIRT6在激光诱导 DNA 损伤时的动态补充。GFP-SIRT6在 WT 细胞 DNA 断裂中迅速招募,但 SIRT1/-细胞中这一过程大部分被推迟(图6E-F) ,这表明 SIRT1在 SIRT6初始招募到 DSBs 中发挥了不可或缺的作用。与此相反,SIRT1对 DSBs 的补充仅仅受到 SIRT6下调的影响,这是由 ChIP-PCR 分析和激光微指针法(图6C-D,g)所确定的。

Figure 6.

Figure 6. 图6SIRT1 facilitates SIRT6 recruitment in DDR. SIRT1促进 SIRT6在复员方案中的招募

(A,B) ChIP-qPCR analysis showing the enrichment of SIRT6 in DSB vicinity in cells treated with si-SIRT1 siRNA or scramble (NC). Immunoblots showing protein levels of FLAG-SIRT6 and SIRT1.

(a,b) ChIP-qPCR 分析显示 SIRT6在 si-SIRT1 siRNA 和 scramble (NC)处理的 DSB 附近细胞中富集。免疫印迹显示 FLAG-SIRT6和 SIRT1蛋白水平。

(C,D) ChIP-qPCR analysis showing the enrichment of SIRT1 in DSB vicinity in cells treated with si-SIRT6 siRNA or NC. Western blots showing protein levels of FLAG-SIRT1 and SIRT6.

(c,d) ChIP-qPCR 分析显示 SIRT1在 si-SIRT6 siRNA 和 NC 处理的 DSB 附近富集。蛋白印迹显示 FLAG-SIRT1和 SIRT6蛋白水平。

(E,F) GFP-SIRT6 was introduced into and Sirt1−/− and Sirt1+/+ MEFs and fluorescence signal was captured after laser damage at various time points. Representative images were shown (E) and relative intensity was calculated by Image J® (F). White dot circles indicate the damage sites. Scale bar, 10 μm.

将(e,f) GFP-SIRT6引入激光损伤后的不同时间点,获得 Sirt1-/-和 Sirt1 +/+ MEFs 信号和荧光信号。用图像 j (f)计算相对强度,并给出具有代表性的图像(e)。白点圆圈表示损害位置。比例尺,10微米。

(G) GFP-SIRT1 were introduced into and Sirt6−/− and Sirt6+/+ MEFs and fluorescence signal was captured after laser damage at various time points. Representative images were shown. Scale bar, 10 μm

(g)在不同时间点引入荧光蛋白 -sirt1,获得 Sirt6-/-和 Sirt6 +/+ MEFs 的荧光信号。展示了具有代表性的图片。比例尺,10微米

We next assessed the function of SIRT6 deacetylation in DNA repair. We found that the acetylation level of SIRT6 was significantly decreased upon CPT treatment, which was abolished in case of SIRT6 K33R or lack of SIRT1 (Figure 7A), implying that SIRT6 is deacetylated by SIRT1 upon DNA damages. We examined the effect of SIRT6 mutants on DNA repair by comet assay, which assesses repair ability at single cell level. To this end, K33R and K33Q were overexpressed in SIRT6−/− cells and DNA repair efficacy was examined. As shown, overexpression of SIRT6 significantly enhanced the DNA repair efficacy upon CPT treatment, while K33Q or H133Y lost the ability. By contrast, K33R promoted DNA repair to an extent comparable to WT (Figure 7B). Together, the data implicate that deacetylation of SIRT6 at K33 is indispensable for DNA repair.

我们接下来评估了 SIRT6脱乙酰基在 DNA 修复中的作用。我们发现,经 CPT 处理后,SIRT6的乙酰化水平显著降低,而 SIRT6 K33R 或缺乏 SIRT1时,SIRT6的乙酰化水平显著降低(图7A) ,提示 SIRT1在 DNA 损伤时对 SIRT6进行脱乙酰化。我们用彗星实验检测了 SIRT6突变体对 DNA 修复的影响,评估了单细胞水平的修复能力。为此,在 SIRT6-/-细胞中高表达 K33R 和 K33Q,并检测 DNA 修复效果。结果表明,过量表达 SIRT6能显著提高 CPT 治疗后 DNA 的修复效果,而 K33Q 和 H133Y 则丧失修复能力。相比之下,K33R 促进 DNA 修复的程度相当于 WT (图7B)。结果表明,在 K33位点,SIRT6的去乙酰化是 DNA 修复不可缺少的环节。

Figure 7.

Figure 7. 图7SIRT6 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

(A) The acetylation level of SIRT6 and K33R in SIRT1+/+ and SIRT1−/− HEK293 cells treated or untreated with CPT (1 μM) for 1 h.

(a)经 CPT (1μM)处理和未处理的 SIRT1 +/+ 和 SIRT1-/-HEK293细胞的 SIRT6和 K33R 的乙酰化水平。

(B) Comet assay in FLAG-SIRT6, K33R, K33Q and HY reconstituted SIRT6 KO cells treated with CPT for 1 h. Data are represented as mean ± s.e.m. **P < 0.01.

(b)经 CPT 处理1h 的 FLAG-SIRT6、 K33R、 K33Q 和 HY 重组 SIRT6细胞的彗星实验结果为: 平均 ± s.e.m. * * p < 0.01。

(C) Comet assay in Sirt1−/− cells transfected with FLAG-SIRT6, K33R, K33Q, HY and SIRT1 and treated with CPT for 1 h. Data are represented as mean ± s.e.m. **P < 0.01.

(c)转染 FLAG-SIRT6、 K33R、 K33Q、 HY 和 SIRT1并经 CPT 处理1h 的 SIRT1-/-细胞的彗星实验结果为平均 ± s.e.m. * p < 0.01。

(D) HR assay in U2OS cells ectopically expressing FLAG-SIRT6, K33R, K33Q or HY. The relative HR value was normalized with vector control. Data are represented as mean ± s.e.m. ***P < 0.001. **P < 0.01.

(d)体外表达 FLAG-SIRT6、 K33R、 K33Q 和 HY 的 U2OS 细胞 HR 分析。采用矢量控制对相对 HR 值进行归一化处理。数据表示为平均 ± s.e.m. * * p < 0.001。* * p < 0.01.

(E) Colony-forming assay in Hela cells ectopically expressing FLAG-SIRT6, K33R or K33Q. Data are represented as mean ± s.e.m. **P < 0.01.

(e)表达 FLAG-SIRT6、 K33R 或 K33Q 的 Hela 细胞体外集落形成实验。数据表示为平均 ± s.e.m * * p < 0.01。

(F) Colony-forming assay in Hella cells stable expressing SIRT6 WT, K33R, K33Q or HY after radiation at indicated dose. Data are represented as mean ± s.e.m. *P < 0.05.

(f) SIRT6 WT、 K33R、 K33Q 和 HY 在海拉细胞中的集落形成实验。数据表示为平均 ± s.e.m. * p < 0.05。

(G) A working model: (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 flanking DSBs. (c) SIRT6 mediates local chromatin remodeling via deacetylating H3K9ac.

(g)一个工作模型: (a) SIRT6在 K33位被 SIRT1脱乙酰,促进 SIRT6的聚合和 dsb 的识别。(b)超过 DSBs,k33脱乙酰基 SIRT6锚定到 γh2ax,并扩展到 DSBs 的局部染色质侧翼。(c) SIRT6通过脱乙酰化 H3K9ac 介导局部染色质重塑。

SIRT1 regulates DNA repair (6). To elucidate the synergistic effects of SIRTs in DNA repair, we examined whether hyper-acetylation of SIRT6 underlines the defective DNA repair in SIRT1−/− cells. As shown, SIRT6, K33R and SIRT1 rescued the defective DNA repair imposed by SIRT1 deficiency, while SIRT6 K33Q and H133Y merely did (Figure 7C). Similar phenomena were observed in Hela cells (Figure S8B-C). HR assay showed that SIRT6 WT and K33R enhanced HR capacity, whereas neither K33Q nor H133Y did (Figure 7D). Further, compared to WT and K33R, K33Q significantly inhibited the colorization of transfected Hela cells (Figure 7E). SIRT6 WT and K33R enhanced cell survival after ionized radiation (Figure 7F).

SIRT1调节 DNA 修复(6)。为了阐明 SIRTs 在 DNA 修复中的协同作用,我们研究了 SIRT6的高乙酰化是否表明 SIRT1-/-细胞 DNA 修复缺陷。如图所示,SIRT6、 K33R 和 SIRT1挽救了 SIRT1缺陷引起的 DNA 修复缺陷,而 SIRT6 K33Q 和 H133Y 仅仅挽救了 DNA 修复缺陷(图7C)。在 Hela 细胞中也观察到类似的现象(图 S8B-C)。HR 实验表明,SIRT6 WT 和 K33R 均能提高 HR 能力,而 K33Q 和 H133Y 均不能提高 HR 能力(图7D)。此外,与 WT 和 K33R 相比,K33Q 显著抑制转染 Hela 细胞的着色(图7E)。SIRT6 WT 和 K33R 增强电离辐射后的细胞存活(图7F)。

Altogether, the above data implicate a synergistic action between SIRT1 and SIRT6 in regulating DDR and DNA repair. We propose a model–SIRT6 is deacetylated by SIRT1 at K33, thus promoting its polymerization and recognition of DSBs; K33-deacetylated SIRT6 anchors to γH2AX, allowing expansion and retention on the chromatin flanking DSBs and subsequent remodeling, likely via deacetylating H3K9ac (Figure 7G).

综上所述,SIRT1和 SIRT6在调节 DDR 和 DNA 修复方面具有协同作用。我们提出了一个模型-SIRT6在 K33上被 SIRT1去乙酰化,从而促进其对 dsb 的聚合和识别; K33去乙酰化 SIRT6锚定到 γh2ax 上,允许扩展和保留在 dsb 的侧翼,并随后重塑,很可能是通过去乙酰化 H3K9ac (图7G)。

Discussion

讨论

DDR is highly orchestrated and initiated by DNA break-sensing (1). The MRN complex (32), Ku complex (28), RPA (33) and PARP1 (34,35) directly recognize DSBs. SIRTs are among the earliest factors that are recruited to DSBs (17,18), facilitating recruitment of PARP1 (7). Consistent with published data (23), we found that SIRT6 oligomerizes and recognizes DSBs via a DSB-binding pocket generated by the N- and a C-termini of two adjacent molecules. This is consistent with a report showing that both N- and C- termini are essential for chromatin association of SIRT6 (26). Recently, using a super-resolution fluorescent particle tracking method, Yang et al found that the binding of PARP1 to DSBs happens earlier than SIRT6 but transient (36). One possible explanation is that, PARP1 is first recruited to DSBs; later-on recruited SIRTs directly by DSBs facilitates the stabilization and expansion of PARP1 in surrounding region.

DDR 是由 DNA 断裂感应(1)高度协调和启动的。MRN 复合物(32)、 Ku 复合物(28)、 RPA (33)和 PARP1(34,35)直接识别 dsb。Sirt 是 DSBs (17,18)招募的最早因素之一,有助于 PARP1(7)的招募。与已发表的数据(23)一致,我们发现 SIRT6通过两个相邻分子的 n-和一个 c- 末端产生的 dsb 结合口袋对 DSBs 进行寡聚和识别。这与一份报告表明 n-和 c-termini 对于 SIRT6(26)的染色质联合是必不可少的一致。最近,Yang 等人利用超分辨率荧光粒子跟踪技术发现 PARP1与 DSBs 的结合早于 SIRT6,但是是短暂的(36)。一个可能的解释是,PARP1首先被招募到 dsb; 后来由 dsb 直接招募的 sirt 有助于 PARP1在周围地区的稳定和扩展。

SIRTs share similar functions in DDR and DNA repair; upon DNA damage, both SIRT1 and SIRT6 are rapidly mobilized to DSBs (7,17,18). SIRT1 redistributes on chromatin and deacetylates XPA, NBS1 and Ku70 to promote DNA repair (811). SIRT6 mono-ribosylates PARP1 to enhance its activity (16), and facilitates subsequent recruitment of SNF2H, H2AX and DNA-PKcs (1215). Here we revealed a synergistic action between nuclear SIRTs–SIRT1 deacetylates SIRT6 to promote its mobilization to DSBs. K33R mutant, mimicking hypo-acetylated SIRT6, rescues DNA repair defects in SIRT1 null cells. Interestingly, phosphorylation of SIRT6 on S10 by JNK promotes subsequent recruitment itself and PARP1 upon oxidative stress, also supporting an essential role of N terminus for DSB-recruitment (15). Consistent with the cooperative action between SIRT1 and SIRT6, independent studies revealed interaction between SIRT1 and SIRT7, showing that SIRT1 recruits SIRT7 to promote cancer cell metastasis (37), and that SIRT1 and SIRT7 antagonistically regulate adipogenesis (38).

SIRTs 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 dsb 中(7,17,18)。SIRT1在染色质和去乙酰化物 XPA、 NBS1和 Ku70上重新分布,促进 DNA 修复(8-11)。SIRT6单核糖基化使 PARP1活性增强(16) ,并促进 SNF2H、 H2AX 和 DNA-PKcs (12-15)的补充。在这里,我们揭示了核 SIRTs-SIRT1脱乙酰基 SIRT6之间的协同作用,以促进其动员到 dsb。模拟低乙酰化 SIRT6的 K33R 突变体对 SIRT1缺失细胞 DNA 修复缺陷的作用。有趣的是,JNK 对 S10蛋白的 SIRT6磷酸化促进了后续的补充作用,而 PARP1则促进了氧化应激的补充作用,也支持了 n 端对 dsb 补充的重要作用(15)。与 SIRT1和 SIRT6的协同作用相一致的是,独立研究揭示了 SIRT1和 SIRT7之间的相互作用,表明 SIRT1能够诱导 SIRT7促进癌细胞转移(37) ,SIRT1和 SIRT7能够拮抗脂肪生成(38)。

The acetylation levels of H3K9 and H3K56 decrease upon DNA damage and then goes back to original level (39). SIRT1 and SIRT6 are the deacetylases of H3K9ac and H3K56ac; both are recruited to DSBs upon DNA damage, indicating that SIRT1 and/or SIRT6 might contribute to the reduced H3K9ac and H3K56ac level. In addition, although mechanistically unclear, the levels of H3K9ac and H3K56ac are negatively correlated with γH2AX. In current study, we found γH2AX is not required for recruiting SIRT6 at the beginning but indispensable for the retention of SIRT6 on local chromatin surrounding DSBs. This is consistent with reports that γH2AX is dispensable for initial reorganization of DNA breaks but rather serves as a platform to stabilize repair factors like NBS1, 53BP1 and BRCA1 (2). SIRT6 might deacetylate H3K9ac and/or H3K56ac surrounding DSBs, bridging γH2AX to chromatin remodeling. Together, the findings provide a scenario how γH2AX and histone modifiers coordinate to amplify DDR.

H3K9和 H3K56的乙酰化水平在 DNA 损伤后下降,然后回到原来的水平(39)。SIRT1和 SIRT6是 H3K9ac 和 H3K56ac 的去乙酰化酶,两者在 DNA 损伤时都被招募到 dsb 中,这表明 SIRT1和/或 SIRT6可能有助于 H3K9ac 和 H3K56ac 水平的降低。H3K9ac 和 H3K56ac 水平与 γh2ax 呈负相关,虽然机制尚不清楚。在目前的研究中,我们发现 γh2ax 并不是最初招募 SIRT6所必需的,但是对于保留 SIRT6在 DSBs 周围的局部染色质上是必不可少的。这与有关 γh2ax 对 DNA 断裂的初始重组不可或缺的报道相一致,而是作为稳定 NBS1、53BP1和 BRCA1(2)等修复因子的平台。SIRT6可能包围着 DSBs 的脱乙酰 H3K9ac 和/或 H3K56ac,将 γh2ax 桥接到染色质重塑。总之,这些发现提供了一个场景,γh2ax 和组蛋白修饰剂如何协同增强 DDR。

SIRT6 together with SNF2H stabilize γH2AX foci (40). Here we found that γH2AX is required to anchor SIRT6 to DSBs, providing a positive feedback regulation between SIRT6 and γH2AX. It is consistent with reports showing a distinct reduction of γH2AX and improper DDR in Sirt6−/− and Sirt1−/− cells. Recent advances suggest electrostatic force between negative charge phosphate group and positive charge lysine as a novel form of protein-protein interaction (27). It is plausible to speculate that (de)acetylation might act as a switch to modulate such interaction between SIRT6 and γH2AX.

SIRT6联合 SNF2H 稳定 γh2ax 区(40)。在这里,我们发现需要 γh2ax 将 SIRT6锚定到 dsb,在 SIRT6和 γh2ax 之间提供一个正反馈调节。这与报告显示的在 Sirt6-/-和 Sirt1-/-细胞中 γh2ax 明显减少和不适当的 DDR 一致。最新进展表明,磷酸负电荷基团和正电荷赖氨酸之间的静电力作为蛋白质-蛋白质相互作用的一种新形式(27)。可以推测(去)乙酰化作用可能是调节 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 identified a direct binding of SIRT6 to DNA breaks, and 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 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与 DNA 断裂的直接结合,以及 SIRT6与 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

物料及方法

Antibodies, Oligos and Plasmids

抗体、寡核苷酸和质粒

Commercial antibodies used in this study includes: SIRT6, SNF2H, pan-AcK, H3 and γH2AX (Abcam), H2AX, SIRT1 and GST (CST), H3K9ac and H3K56ac (Millipore), SIRT6 (Novus), Tubulin and FLAG (Sigma).

本研究使用的商用抗体包括: SIRT6、 SNF2H、 pan-AcK、 H3和 γH2AX (Abcam)、 H2AX、 SIRT1和 GST (CST)、 H3K9ac 和 H3K56ac (Millipore)、 SIRT6(Novus)、微管蛋白和 FLAG (Sigma)。

Oligos used for RNA interference:

用于 RNA干扰的 Oligos:

  • siSIRT6, 5’-AAGAAUGUGCCAAGUGUAAGA-3’;siSIRT6,5’-aagaaugugcaaguaaga-3’ ;
  • isSIRT1, 5’-ACUUUGCUGUAACCCUGUA-3’.isSIRT1,5’-acuugcuaccugua-3’。

Primers used for ChIP qPCR:

芯片 qPCR 的引物:

  • I-SceI-2k-F, 5’-GCCCATATATGGAGTTCCGC-3’;I-se-2k-f,5’-gcccatatggttccgc-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’。

gRNA used for CRISPR/Cas9 gene editing:

gRNA 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’。
  • gRNA-F, 5’-CACCGATAGCAAGCGGTTCATCAGC-3’gRNA-F,5’-caccgatagagggttcatcagc-3’

Human SIRT6 was cloned into pCDNA3.1 with FLAG; 3×FLAG-SIRT1 was ordered from Addgene. SIRT6ΔC and ΔN were amplified with specific primers and cloned into pKH3HA and pGex vector. 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 directed mutagenesis described below in detail.

人 SIRT6基因与 FLAG 一起被克隆到 pCDNA3.1中,3 × FLAG-sirt1基因在 Addgene 定位。利用特异性引物扩增 SIRT6ΔC 和 δn 基因,克隆到 pKH3HA 和 pGex 载体中。通过定向诱变将 SIRT6赖氨酸33转化为精氨酸(KR) ,或将其转化为谷氨酰胺(KQ)和133组氨酸(HY) ,获得了 KR、 KQ 和 HY 突变体。

Site-directed mutagenesis

定点突变

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 manufactory’s instruction. The PCR product was digested by DpnI endonuclease for 1h at 37°C, followed by 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。根据生产厂家的要求,利用安捷伦公司的火球菌DNA聚合酶和300ng 质粒模板进行诱变。PCR 产物经核酸内切酶酶切,37 ° c 处理1h,进行转化和测序。

Primer used for generation of SIRT6 KR, KQ and HY mutants:

用于 SIRT6 KR、 KQ 和 HY 突变体生成的引物:

  • KR forward: 5‘-GGAGCTGGAGCGGAGGGTGTGGGAACT-3’5‘-ggagctggggggggtggggaact-3’
  • KR reverse: 5‘-AGTTCCCACACCCTCCGCTCCAGCTCC-3’KR 反向: 5‘-agttccaccctgctcccccc-3’
  • KQ forward: 5‘-GGAGCTGGAGCGGCAGGTGTGGGAACT-3’KQ 前锋: 5‘-ggagctgggggggggggggggggggaactt-3’
  • KQ reverse: 5‘-AGTTCCCACACCTGCCGCTCCAGCTCC-3’KQ 反向: 5‘-agttccccacctcccgctcccccc-3’
  • HY forward: 5‘-ACAAACTGGCAGAGCTCTACGGGAACATGTTTGTG-3’5‘-acaactggcagctctacgggaacatgttgg-3’
  • HY reverse: 5‘-CACAAACATGTTCCCGTAGAGCTCTGCCAGTTTGT-3’5’-cacaacatgtcctgccgccgttgt-3’

Immunoprecipitation

免疫沉淀法

HEK293T cells were transfected with indicated plasmids using Lipofetamine®3000 (Invitrogen, USA), according to the manufacturer’s instructions. Cells were lysed 48 h post-transfection in lysis buffer (50 mM Tris-HCl, pH 7.4, 200 mM NaCl, 0.2% NP40, 10% glycerol, 1mM NaF, 1 mM Sodium butyrate, 10 mM Nicotinamide and complete protease inhibitor cocktail, Roche). The cell extracts were incubated with anti-FLAG M2 monoclonal antibody-conjugated agarose beads (Sigma) at 4°C overnight. The immunoprecipitates were boiled with 2×laemmli buffer and were analyzed by Western blotting.

根据生产商的说明书,用 Lipofetamine 3000(美国 Invitrogen)转染 HEK293T 细胞,获得指定的质粒。细胞裂解后48h 在溶解液中(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,罗氏)。细胞提取物与抗 flag M2单克隆抗体偶联的琼脂糖珠(Sigma)在4 ° c 条件下孵育过夜。免疫沉淀物用2 × laemmli 缓冲液煮沸,用西方墨点法分析法进行分析。

Chromatin Immunoprecipitation (ChIP)

染色质免疫沉淀

I-SceI-GR assays were performed as described (30). Hela cells stable transfected with DR-GFP were transiently transfected with RFP-I-SceI-GR together with FLAG-SIRT6, KR, KQ or HY. 48 h after transfection, the cells were treated with 10−7 M of triamcinolone acetonide (TA, Sangon, Shanghai) for 20 min, and fixed to crosslink chromatin with 1% paraformaldehyde at 37°C for 10 min and stopped with 0.125 M glycine. The chromatin was sonicated to 200bps~600bps and incubated with indicated antibodies. After de-cross linking, ChIP-associated DNA were extracted and examined by quantitative real-time PCR.

I-SceI-GR 检测按照描述(30)进行。将稳定转染 DR-GFP 的 Hela 细胞与 FLAG-SIRT6、 KR、 KQ 或 HY 共同瞬时转染 RFP-I-SceI-GR。转染后48h,用10-7m 曲安奈德(Sangon)处理细胞20min,在37 ° c 固定1% 多聚甲醛交联染色质10min,停用0.125 m 甘氨酸。染色质经超声波处理至200bps ~ 600bps,并与特异性抗体共同孵育。去交联后,提取 chip 相关 DNA,用实时荧光定量 PCR 检测。

Comet assay

彗星试验

Comet assay was performed as described (41). Briefly, after CPT treatment, cells were digested into single cell suspension, mixed with 1% agarose at the density of 1 × 105, coated on the slide and followed by incubating in lysis buffer (2% sarkosyl, 0.5M Na2EDTA, 0.5 mg/ml proteinase K) overnight. Slides were incubated with N2 buffer (90 mM Tris, 90 mM boric acid and 2 mM Na2EDTA) and transferred to electrophoresis for 25 min at 0.6 V/cm. Slides were incubated in staining solution containing 2.5 μg/ml of Propidium iodide for 30 min. Images were captured under fluorescent microscope.

彗星实验进行了描述(41)。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,然后在溶解液(2% sarkosyl,0.5 m Na2EDTA,0.5 mg/ml 蛋白酶 k)中培养过夜。用 N2缓冲液(90mm Tris,90mm 硼酸和2mm Na2EDTA)培养载玻片,0.6 V/cm 电泳25min。载玻片在含2.5 μg/ml 碘化丙啶的染色液中培养30min。图像在荧光显微镜下捕获。

Cell fractionation

细胞分级

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

刮细胞,用冷的 PBS 洗涤。用10mm HEPES,10mm KCl,1.5 mM MgCl2,0.34 m 蔗糖,10% 甘油,1mM DTT,0.1% TrionX-100细胞核溶解液悬浮颗粒在冰上放置10分钟,以1300克速度离心10分钟。以3mm EDTA、0.2 mM EGTA、1mm DTT 溶解液悬浮颗粒10min,以1700g 速度离心10min。颗粒作为染色质部分保存。

Micro-point laser irradiation and microscopy

微点激光照射与显微技术

U2OS or MEF cells were seeded on a dish with thin glass bottom (NEST), then locally irradiated with a 365 nm pulsed UV laser (16 Hz pulse, 56% laser output), generated by the micro-point 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 with time-lapse imaging every 20 s for 10 min. Quantification of fluorescence intensity at every time-point was measured by Fiji (image J) software.

将 U2OS 或 MEF 细胞种植在玻璃底的培养皿上,然后利用微点激光照射和烧蚀系统(Andor,power supply TPES24-T120MM,Laser NL100,24V 50W)产生的365nm 脉冲紫外激光(16hz 脉冲,56% 激光输出) ,与尼康 A1共焦成像系统(TuCam)的荧光路径耦合,局部照射 U2OS 或 MEF 细胞。荧光蛋白的补充和保留每20s 连续监测10min。荧光强度的量化在每个时间点用 Fiji (图像 j)软件测量。

CRISPR/Cas9-mediated gene editing

Crispr/cas9介导的基因编辑

CRISPR/Cas9-mediated gene editing was conducted as described (Ran et al.,2013). Briefly, pX459 vector (Addgene#48139) was digested with BbsI and ligated with annealed oligonucleotides. The constructs containing target gRNAs were transfected into HEK293T cells with Lipofetamine3000® (Invitrogen). Cells were selected for 5 days with puromycin 24 h after transfection. Single clone was 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。转染后24h 用嘌呤霉素筛选细胞5d。选择单个克隆进行测序。

Peptide pulldown

肽下拉

The C terminus of H2AX (BGKKATQASQEY) and γH2AX (BGKKATQApSQEY) were synthesized and conjugated with biotin (GL Biochem, Shanghai). For one reaction, 1 μg biotinylated peptides were 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 peptide and protein complexes for 1 h at 4°C, followed by Western blotting.

合成了 H2AX (BGKKATQASQEY)和 γH2AX (BGKKATQApSQEY)的 c 端,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 的生物素化肽。链霉亲和素 Sepharose 珠(GE)用于将多肽和蛋白质复合物在4 ° c 下拉1小时,然后是西方墨点法。

Immunofluorescence staining

免疫荧光染色

Cells were washed with PBS and fixed with 4% formaldehyde for 20 min, followed by permeabilization with cold methanol (−20°C) for 5 min, blocking with 5% BSA for 30 min, incubation 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 h and secondary antibodies (donkey anti-rabbit IgG Alexa Fluor 594 and donkey anti-mouse IgG FITC from Invitrogen, 1:500 dilution in1% BSA) for 1h at room temperature. Cells were then co-stained with DAPI (Invitrogen) and observed under a fluorescent microscope.

用 PBS 洗涤细胞,用4% 甲醛固定20min,然后用冷甲醇(- 20 ° c)渗透5min,用5% BSA 封闭30min,用一级抗体(SIRT1,1:200稀释1% BSA; γh2ax,1:500稀释1% BSA; SIRT6,1:200稀释1% BSA)孵育1h,室温下用二级抗体(抗兔 IgG 抗兔血清 Fluor 594和抗兔 IgG 抗体 FITC,1:500稀释1% BSA)孵育1h。然后与 DAPI (Invitrogen)共染,在荧光显微镜下观察细胞形态。

HR assay

心率测定

U2OS cells stabled transfected with DR-GFP were transfected with HA-I-SceI together with Flag-SIRT6 WT, K33R, K33Q or HY respectively. After transfection for 48h, cells were harvested and analyzed the GFP positive cell ratio per 104 cells by flow cytometry (BD). Relative HR efficiency was normalized with vector control.

将 HA-I-SceI 与 Flag-SIRT6 WT、 K33R、 K33Q、 HY 分别转染稳定转染 DR-GFP 的 U2OS 细胞。转染48h 后,采集细胞,用流式细胞仪(BD)分析 GFP 阳性细胞率。采用矢量控制对相对 HR 效率进行了归一化。

Colony formation assay

集落形成试验

Hella cells were seeded into six-well plates 24 hours after transfection in defined numbers. 24 hours following re-plating the cells were dosed with increased amounts of radiation. Fresh media was added after seven days. Once reached 50 cells in size (10-14 days), colonies were fixed with 20% methanol, and stained with crystal violet. Colonies (>50 cells) were used for analysis. Ionizing radiation was delivered by an X-Rad 320 irradiator (Precision X-Ray Inc. N. Branford, CT, USA).

定量转染后24小时将 Hella 细胞种植到6孔板中。重新电镀24小时后,电池被施以更多的辐射剂量。七天后添加新鲜培养基。一旦细胞体积达到50个(10-14天) ,用20% 甲醇固定菌落,并用结晶紫染色。菌落(> 50个细胞)进行分析。电离辐射是由 X-Rad 320辐射仪(Precision x- 射线公司)发射的。布兰福德,康涅狄格州,美国)。

DNA pulldown assay

脱氧核糖核酸下降试验

DNA binding assay was performed following previous report (42). Briefly, biotin conjugated DNA duplex with the size of 220bp was generated by PCR amplification using biotin-labeled primers and I-sceI plasmid as template. In regard of DNA pulldown assay, 10pmol biotinylated DNA duplex were incublated with 0.5μg 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 hour. The beads were then collected and washed with binding buffer for 3 times. The beads were subsequently boiled in 2×laemmli buffer and analyzed by Western blot. For linear and circular DNA competition assay, the ratios of non-biotin labeled linear/circular DNA to biotin DNA duplex were 5:1 or 10:1. Linear DNA were generated with PCR amplification using non-biotin-labeled primers, and circular DNA were obtained by cloning PCR product into pCDNA 3.1 plasmid.

DNA 结合试验是在先前的报告(42)之后进行的。以生物素标记的引物和 i ー scei 质粒为模板,通过 PCR 扩增得到大小为220bp 的生物素结合 DNA 双链。用0.5 μg 表示重组蛋白的10pmol 生物素化 DNA 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 条件下连夜浸泡。链霉亲和素 Sepharose 珠子(GE)被添加到第二天,并孵化另一个1小时。收集珠子,用装订缓冲液洗涤3次。然后在2 × laemmli 缓冲液中煮沸,用 Western blot 分析。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 的比值分别为5:1和10:1。采用非生物素标记引物进行 PCR 扩增,获得线性 DNA,将 PCR 产物克隆到 pCDNA 3.1质粒中,获得圆形 DNA。

The sequences used for PCR:

用于 PCR 的序列:

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

Fluorescence polarization assay

荧光偏振分析

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

在体外纯化 SIRT1、 SIRT6和 SIRT7重组蛋白,并与 FAM 结合的双链 DNA (20nm)在冰上孵育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法。

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