Mutations in the ubiquitin binding UBZ motif of DNA polymerase η do not impair its function in translesion synthesis during replication
Mutations in the ubiquitin binding UBZ motif of DNA polymerase η do not impair its function in translesion synthesis during replication
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DOI：
10.1128/mcb.01196-07
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发表时间：
2007-10-01
影响因子：
5.3
通讯作者：
Prakash, Satya
中科院分区：
文献类型：
--
作者：
Acharya, Narottam;Brahma, Amrita;Prakash, Satya
用DNA损伤剂处理酿酒酵母细胞，通过Rad 6-Rad 18使赖氨酸164连接的PCNA单泛素化。近年来，在跨损伤合成（translesion synthesis，TLS）DNA聚合酶中发现了大量的泛素（ubiquitin，Ub）结合域（ubiquitin binding domain，UBD），并提出TLS聚合酶中的UBD影响其与PCNA上Ub的结合，这种结合模式是TLS聚合酶在停滞复制叉位点接近PCNA所必需的。为了评估UBD与TLS中PCNA上的Ub部分结合的贡献，我们研究了C、H、锌结合基序和位于酵母Pol eta的UBZ结构域的α-螺旋部分的保守D570残基中的突变的影响。我们发现C2 H2基序的突变对UV敏感性或UV诱变没有明显的影响，而D570残基的突变对Pol eta功能有不利影响。Poleta与PCNA或Ub-PCNA刺激DNA合成不受C，H，基序或D570残基突变的影响。这些观察结果使我们认为，Ub通过其UBZ结构域对PCNA的结合不是聚合酶eta在复制过程中在TLS中发挥作用的能力的必要条件。
Treatment of Saccharomyces cerevisiae cells with DNA-damaging agents elicits lysine 164-linked PCNA monoubiquitination by Rad6-Rad18. Recently, a number of ubiquitin (Ub) binding domains (UBDs) have been identified in translesion synthesis (TLS) DNA polymerases and it has been proposed that the UBD in a TLS polymerase affects its binding to Ub on PCNA and that this binding mode is indispensable for a TLS polymerase to access PCNA at the site of a stalled replication fork. To evaluate the contribution of the binding of UBDs to the Ub moiety on PCNA in TLS, we have examined the effects of mutations in the C,H, zinc binding motif and in the conserved D570 residue that lies in the a-helix portion of the UBZ domain of yeast Pol eta. We find that mutations in the C2H2 motif have no perceptible effect on UV sensitivity or UV mutagenesis, whereas a mutation of the D570 residue adversely affects Pol eta function. The stimulation of DNA synthesis by Pol eta with PCNA or Ub-PCNA was not affected by mutations in the C,H, motif or the D570 residue. These observations lead us to suggest that the binding of Ub on PCNA via its UBZ domain is not a necessary requirement for the ability of polymerase eta to function in TLS during replication.