Hwang Bor-Jang, Jin Jin, Gao Ying, Shi Guoli, Madabushi Amrita, Yan Austin, Guan Xin, Zalzman Michal, Nakajima Satoshi, Lan Li, Lu A-Lien
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD, 21201, USA.
University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, 5117 Centre Avenue, Pittsburgh, PA, 15213, USA.
BMC Mol Biol. 2015 Jun 11;16:12. doi: 10.1186/s12867-015-0041-9.
SIRT6, a member of the NAD(+)-dependent histone/protein deacetylase family, regulates genomic stability, metabolism, and lifespan. MYH glycosylase and APE1 are two base excision repair (BER) enzymes involved in mutation avoidance from oxidative DNA damage. Rad9-Rad1-Hus1 (9-1-1) checkpoint clamp promotes cell cycle checkpoint signaling and DNA repair. BER is coordinated with the checkpoint machinery and requires chromatin remodeling for efficient repair. SIRT6 is involved in DNA double-strand break repair and has been implicated in BER. Here we investigate the direct physical and functional interactions between SIRT6 and BER enzymes.
We show that SIRT6 interacts with and stimulates MYH glycosylase and APE1. In addition, SIRT6 interacts with the 9-1-1 checkpoint clamp. These interactions are enhanced following oxidative stress. The interdomain connector of MYH is important for interactions with SIRT6, APE1, and 9-1-1. Mutagenesis studies indicate that SIRT6, APE1, and Hus1 bind overlapping but different sequence motifs on MYH. However, there is no competition of APE1, Hus1, or SIRT6 binding to MYH. Rather, one MYH partner enhances the association of the other two partners to MYH. Moreover, APE1 and Hus1 act together to stabilize the MYH/SIRT6 complex. Within human cells, MYH and SIRT6 are efficiently recruited to confined oxidative DNA damage sites within transcriptionally active chromatin, but not within repressive chromatin. In addition, Myh foci induced by oxidative stress and Sirt6 depletion are frequently localized on mouse telomeres.
Although SIRT6, APE1, and 9-1-1 bind to the interdomain connector of MYH, they do not compete for MYH association. Our findings indicate that SIRT6 forms a complex with MYH, APE1, and 9-1-1 to maintain genomic and telomeric integrity in mammalian cells.
SIRT6是烟酰胺腺嘌呤二核苷酸（NAD⁺）依赖性组蛋白/蛋白质脱乙酰酶家族的成员，可调节基因组稳定性、代谢和寿命。MYH糖基化酶和APE1是两种碱基切除修复（BER）酶，参与避免氧化DNA损伤引起的突变。Rad9-Rad1-Hus1（9-1-1）检查点钳促进细胞周期检查点信号传导和DNA修复。BER与检查点机制相互协调，并且需要染色质重塑以实现有效修复。SIRT6参与DNA双链断裂修复，并与BER有关。在这里，我们研究SIRT6与BER酶之间直接的物理和功能相互作用。
我们发现SIRT6与MYH糖基化酶和APE1相互作用并刺激它们。此外，SIRT6与9-1-1检查点钳相互作用。氧化应激后，这些相互作用增强。MYH的结构域间连接体对于与SIRT6、APE1和9-1-1的相互作用很重要。诱变研究表明，SIRT6、APE1和Hus1在MYH上结合重叠但不同的序列基序。然而，APE1、Hus1或SIRT6与MYH的结合不存在竞争。相反，一个MYH结合伙伴会增强其他两个伙伴与MYH的结合。此外，APE1和Hus1共同作用以稳定MYH/SIRT6复合物。在人类细胞中，MYH和SIRT6被有效地募集到转录活跃染色质内的局限性氧化DNA损伤位点，但不会募集到抑制性染色质内。此外，氧化应激和Sirt6缺失诱导的Myh病灶经常定位在小鼠端粒上。
尽管SIRT6、APE1和9-1-1与MYH的结构域间连接体结合，但它们并不竞争与MYH的结合。我们的研究结果表明，SIRT6与MYH、APE1和9-1-1形成复合物，以维持哺乳动物细胞中的基因组和端粒完整性。
