ALC1/CHD1L, a chromatin-remodeling enzyme, is required for efficient base excision repair.
ALC1/CHD1L, a chromatin-remodeling enzyme, is required for efficient base excision repair.
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DOI：
10.1371/journal.pone.0188320
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Hirota K
中科院分区：
文献类型：
--
作者：
Tsuda M;Cho K;Ooka M;Shimizu N;Watanabe R;Yasui A;Nakazawa Y;Ogi T;Harada H;Agama K;Nakamura J;Asada R;Fujiike H;Sakuma T;Yamamoto T;Murai J;Hiraoka M;Koike K;Pommier Y;Takeda S;Hirota K
ALC1/CHD1L是SNF2超家族ATPase的成员之一，其携带的大结构域可与聚(ADP-核糖)结合。聚(ADP-核糖)聚合酶(PARP)1和2在DNA链裂解位点合成聚ADP-核糖，促进碱基切除修复(BER)。虽然ALC1的缺失导致对各种DNA损伤剂(H_2O_2、紫外线和平阳霉素)的敏感性增加，但ALC_1在误码率中所起的作用尚未确定。为了探讨这一作用，以及ALC1ATPase活性在误码率中的作用，我们在不表达PARP2的鸡DT40细胞中阻断了ALC1ATPase-DEAD(E165Q)突变，并将其插入到ALC1ATPase基因中。由此产生的ALc1-/-和ALc1-/E165Q细胞对烷化剂甲基甲烷磺酸盐(MMS)和H_2O_2表现出难以区分的超敏反应，表明ATPase在DNA损伤反应中发挥重要作用。PARP1-/-和ALC1-/-/PARP1-/-细胞对MMS的敏感性非常相似，表明ALC1和PARP1在误码率中协同作用。在脉冲暴露于过氧化氢后，PARP1-/-和ALC1-/-/PARP1-/-细胞在修复单链断裂方面表现出类似的延迟动力学，单链断裂是作为BER中间体出现的。为了确定alc1‘S在哺乳动物细胞误码率中的作用，我们在人TK6细胞中阻断了alc1基因。在MMS和H_2O_2作用下，ALC1-/-TK6细胞的单链断裂修复出现延迟。因此，我们得出结论：ALC1在误码率中起一定作用。暴露于H_2O_2后，ALC_1-/-细胞染色质松弛受损。因此，我们认为ALC1是一种独特的BER因子，在染色质环境中发挥作用，很可能是作为染色质重塑酶发挥作用。
ALC1/CHD1L is a member of the SNF2 superfamily of ATPases carrying a macrodomain that binds poly(ADP-ribose). Poly(ADP-ribose) polymerase (PARP) 1 and 2 synthesize poly(ADP-ribose) at DNA-strand cleavage sites, promoting base excision repair (BER). Although depletion of ALC1 causes increased sensitivity to various DNA-damaging agents (H2O2, UV, and phleomycin), the role played by ALC1 in BER has not yet been established. To explore this role, as well as the role of ALC1’s ATPase activity in BER, we disrupted the ALC1 gene and inserted the ATPase-dead (E165Q) mutation into the ALC1 gene in chicken DT40 cells, which do not express PARP2. The resulting ALC1-/- and ALC1-/E165Q cells displayed an indistinguishable hypersensitivity to methylmethane sulfonate (MMS), an alkylating agent, and to H2O2, indicating that ATPase plays an essential role in the DNA-damage response. PARP1-/- and ALC1-/-/PARP1-/- cells exhibited a very similar sensitivity to MMS, suggesting that ALC1 and PARP1 collaborate in BER. Following pulse-exposure to H2O2, PARP1-/- and ALC1-/-/PARP1-/- cells showed similarly delayed kinetics in the repair of single-strand breaks, which arise as BER intermediates. To ascertain ALC1’s role in BER in mammalian cells, we disrupted the ALC1 gene in human TK6 cells. Following exposure to MMS and to H2O2, the ALC1-/- TK6 cell line showed a delay in single-strand-break repair. We therefore conclude that ALC1 plays a role in BER. Following exposure to H2O2, ALC1-/- cells showed compromised chromatin relaxation. We thus propose that ALC1 is a unique BER factor that functions in a chromatin context, most likely as a chromatin-remodeling enzyme.
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