PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells.
PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells.
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DOI：
10.1371/journal.pgen.1002262
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发表时间：
2011-09
期刊：
影响因子：
4.5
通讯作者：
Livneh Z
中科院分区：
文献类型：
--
作者：
Hendel A;Krijger PH;Diamant N;Goren Z;Langerak P;Kim J;Reissner T;Lee KY;Geacintov NE;Carell T;Myung K;Tateishi S;D'Andrea A;Jacobs H;Livneh Z
翻译DNA合成（TLS）是一种DNA损伤耐受机制，其中特化的低保真DNA聚合酶绕过复制阻断病变，通常与诱变有关。在酿酒酵母中，TLS的一个关键事件是PCNA的单泛素化，这使得特异性聚合酶能够通过泛素结合域招募到受损位点。然而，在哺乳动物中，关于TLS是否需要泛素化PCNA （PCNA- ub）存在争议。我们发现，与PCNA +/+细胞相比，紫外光诱导的单链DNA （ssDNA）区域在PcnaK164R/K164R细胞中积累更快，消失更慢，这与TLS缺陷一致，这些细胞对PCNA泛素化具有抗性。使用具有位点特异性病变的间隙质粒对这些细胞中的TLS进行直接分析，表明TLS在紫外线病变和顺铂诱导的链内GG交联中强烈减少。在缺乏能使PCNA单泛素化的E3泛素连接酶Rad18的细胞中也获得了类似的效果。一致地，缺乏Usp1（去泛素化PCNA的酶）的细胞在紫外线损伤和顺铂加合物上表现出增加的TLS。相反，缺乏多泛素化PCNA的rad5同系物Shprh和Hltf的细胞表现出正常的TLS。在PcnaK164R/K164R小鼠胚胎成纤维细胞中敲除TLS基因Rev3L、PolH或Rev1的表达，导致它们对紫外线辐射的敏感性增加，表明存在独立于PCNA-Ub的TLS途径。综上所述，这些结果表明PCNA-Ub是最大TLS所必需的。然而，TLS聚合酶也可以在缺乏PCNA-Ub的情况下被募集到受损的DNA上，并执行TLS，尽管效率显著降低，致突变特异性也发生了改变。DNA损伤会阻碍复制，导致突变、基因组不稳定和癌症。在不可能去除DNA损伤并在复制之前恢复原始序列的情况下，细胞利用DNA损伤耐受机制，帮助复制绕过病变。一个主要的普遍耐受机制是翻译DNA合成（TLS），其中专门的低保真DNA聚合酶延长DNA穿过病变。这是一把双刃剑，因为完成复制的代价是增加了与病变相反的点突变的风险。因此，TLS调控对于防止突变率的上升至关重要。TLS调控的一个关键因素是一种叫做泛素的小蛋白质附着在PCNA蛋白上，这是一种滑动的DNA夹，将DNA聚合酶绑在DNA上，其功能是将TLS DNA聚合酶招募到DNA中的受损部位。虽然在酵母中，PCNA的这种修饰对TLS至关重要，但关于其在哺乳动物中的重要性存在争议。本研究表明，在哺乳动物细胞中，泛素对PCNA的修饰是重要的，但存在次要但重要的TLS机制，这些机制在泛素缺失的情况下起作用，并导致突变结果的改变。
Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in PcnaK164R/K164R cells, which are resistant to PCNA ubiquitination, compared to Pcna+/+ cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in PcnaK164R/K164R mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity. DNA damage can block replication and lead to mutations, genomic instability, and cancer. In cases when the removal of DNA damage and restoration of the original sequence prior to replication is impossible, cells utilize DNA damage tolerance mechanisms, which help replication to bypass the lesions. A major universal tolerance mechanism is translesion DNA synthesis (TLS), in which specialized low-fidelity DNA polymerases elongate the DNA across the lesion. This is a double-edged sword because the price of completing replication is an increased risk of point mutations opposite the lesion. Thus, TLS regulation is critical for preventing an escalation in mutation rates. A key element in TLS regulation is the attachment of a small protein called ubiquitin to the PCNA protein, a sliding DNA clamp that tethers the DNA polymerases to DNA, which functions to recruit the TLS DNA polymerase to the damaged site in DNA. While in yeast this modification of PCNA is crucial for TLS, there is a debate about its importance in mammals. Here we show that in mammalian cells the modification of PCNA by ubiquitin is important, but there exist secondary yet significant TLS mechanisms that operate in its absence and have an altered mutational outcome.
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影响因子：
15.3
作者：
Langerak, Petra;Nygren, Anders O. H.;Krijger, Peter H. L.;van den Berk, Paul C. M.;Jacobs, Heinz
通讯作者：
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DOI：
10.1007/978-1-4419-6676-6_15
发表时间：
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期刊：
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影响因子：
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通讯作者：
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