Kehrli Keffy, Phelps Michael, Lazarchuk Pavlo, Chen Eleanor, Monnat Ray, Sidorova Julia M
From the Department of Pathology and.
From the Department of Pathology and; Department of Genome Sciences, University of Washington, Seattle, Washington 98195.
J Biol Chem. 2016 Nov 18;291(47):24487-24503. doi: 10.1074/jbc.M115.708594. Epub 2016 Sep 26.
The WRN helicase/exonuclease is mutated in Werner syndrome of genomic instability and premature aging. WRN-depleted fibroblasts, although remaining largely viable, have a reduced capacity to maintain replication forks active during a transient hydroxyurea-induced arrest. A strand exchange protein, RAD51, is also required for replication fork maintenance, and here we show that recruitment of RAD51 to stalled forks is reduced in the absence of WRN. We performed a siRNA screen for genes that are required for viability of WRN-depleted cells after hydroxyurea treatment, and identified HDAC1, a member of the class I histone deacetylase family. One of the functions of HDAC1, which it performs together with a close homolog HDAC2, is deacetylation of new histone H4 deposited at replication forks. We show that HDAC1 depletion exacerbates defects in fork reactivation and progression after hydroxyurea treatment observed in WRN- or RAD51-deficient cells. The additive WRN, HDAC1 loss-of-function phenotype is also observed with a catalytic mutant of HDAC1; however, it does not correlate with changes in histone H4 deacetylation at replication forks. On the other hand, inhibition of histone deacetylation by an inhibitor specific to HDACs 1-3, CI-994, correlates with increased processing of newly synthesized DNA strands in hydroxyurea-stalled forks. WRN co-precipitates with HDAC1 and HDAC2. Taken together, our findings indicate that WRN interacts with HDACs 1 and 2 to facilitate activity of stalled replication forks under conditions of replication stress.
WRN解旋酶/核酸外切酶在基因组不稳定和早衰的沃纳综合征中发生突变。WRN缺失的成纤维细胞虽然在很大程度上仍能存活，但在短暂的羟基脲诱导的停滞期间，维持复制叉活跃的能力有所下降。一种链交换蛋白RAD51也是维持复制叉所必需的，我们在此表明，在没有WRN的情况下，RAD51募集到停滞叉的能力会降低。我们对羟基脲处理后WRN缺失细胞存活所需的基因进行了siRNA筛选，并鉴定出I类组蛋白脱乙酰酶家族成员HDAC1。HDAC1与其紧密同源物HDAC2共同执行的功能之一是对沉积在复制叉处的新组蛋白H4进行脱乙酰化。我们表明，HDAC1缺失会加剧在WRN或RAD51缺陷细胞中观察到的羟基脲处理后叉重新激活和进展的缺陷。HDAC1的催化突变体也观察到了WRN、HDAC1功能丧失的累加表型；然而，它与复制叉处组蛋白H4脱乙酰化的变化无关。另一方面，HDACs 1-3特异性抑制剂CI-994对组蛋白脱乙酰化的抑制与羟基脲停滞叉中新合成DNA链的加工增加相关。WRN与HDAC1和HDAC2共沉淀。综上所述，我们的研究结果表明，在复制应激条件下，WRN与HDAC1和HDAC2相互作用以促进停滞复制叉的活性。
