The translesion DNA polymerases Pol ζ and Rev1 are activated independently of PCNA ubiquitination upon UV radiation in mutants of DNA polymerase δ.
The translesion DNA polymerases Pol ζ and Rev1 are activated independently of PCNA ubiquitination upon UV radiation in mutants of DNA polymerase δ.
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DOI：
10.1371/journal.pgen.1007119
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发表时间：
2017-12
期刊：
影响因子：
4.5
通讯作者：
Maloisel L
中科院分区：
文献类型：
--
作者：
Tellier-Lebegue C;Dizet E;Ma E;Veaute X;Coïc E;Charbonnier JB;Maloisel L
复制型DNA聚合酶不能在碱基损伤部位有效地插入核苷酸。这一功能由专门的跨损伤DNA合成(TLS)聚合酶接管，以允许在DNA损伤存在的情况下完成DNA复制。在真核生物中，RAD6和RAD18介导的增殖细胞核抗原赖氨酸164泛素化促进TLS聚合酶的募集，使细胞能够有效地应对DNA损伤。然而，一些研究表明，在没有增殖细胞核抗原泛素化的情况下，也可以招募TLS聚合酶。我们推测，聚合酶δ(POLδ)亚单位之间和/或POLδ与增殖细胞核抗原在引物/模板连接处的相互作用的稳定性是决定增殖细胞核抗原泛素化要求的关键因素。为了验证这一假设，我们使用了Pol3和Pol31之间的相互作用被抑制的POLδ的结构突变体。我们发现在酵母中，与RAD18Δ相关的紫外线超敏反应被Pol3-ct抑制，pol3-ct是POL3基因的一个突变等位基因，编码复制的POLδ的催化亚基。Pol3-CT的抑制作用依赖于REV1和POLζTLS聚合酶。这一结果有力地表明，当PolLδ与增殖细胞核抗原的相互作用因突变而部分受损时，TLS聚合酶对增殖细胞核抗原泛素化的依赖程度可能要低得多。与这个模型一致，我们发现Pol3-FI等位基因抑制了RAD18Δ相关的紫外线敏感性，就像观察到的Pol3-CT一样。这个POL3等位基因在一个假定的增殖细胞核抗原相互作用多肽(PIP)基序中携带突变。然后，我们提供了分子和遗传学证据，证明这个基序可以间接地促进POLδ-增殖细胞核抗原的相互作用，尽管它不是真正的PIP。总体而言，我们的结果表明，增殖细胞核抗原泛素化的主要作用是允许TLS聚合酶在δ损伤时竞争Pol DNA。在每个细胞周期的S期，复制型DNA聚合酶具有复制基因组DNA的重要作用。如果要复制的DNA没有损坏，DNA复制就会顺利而准确地进行。相反，当复制型DNA聚合酶遇到模板上的碱基受损时，它们会突然停滞。在这种情况下，另一种特殊的DNA聚合酶被招募来在碱基损伤的位置插入核苷酸。然而，这些跨病变聚合酶不是进行性的，它们的准确性很差。因此，它们需要受到严格的监管。这是通过小泛素多肽与聚合酶辅助因子增殖细胞核抗原的共价结合实现的，该结合随后在DNA损伤部位触发跨损伤聚合酶的募集。然而，独立于增殖细胞核抗原泛素化的跨病变聚合酶的募集也已被证明，尽管潜在的机制尚不清楚。此外，这一观察结果使人们更难理解增殖细胞核抗原泛素化的确切作用。在这里，我们在酿酒酵母中提出了强有力的遗传证据，暗示复制型δ聚合酶(PolDNA聚合酶δ)可以防止紫外线照射后跨损伤聚合酶POLζ和Rev1的募集，除非增殖细胞核抗原被泛素化。因此，增殖细胞核抗原泛素化的主要作用将是允许跨损伤聚合酶在DNA损伤时竞争Polδ。此外，我们的结果使我们提出，当Polδ与增殖细胞核抗原的关联受到挑战时，例如在难以复制的基因座上，可以独立于增殖细胞核抗原泛素化招募跨损伤聚合酶。
Replicative DNA polymerases cannot insert efficiently nucleotides at sites of base lesions. This function is taken over by specialized translesion DNA synthesis (TLS) polymerases to allow DNA replication completion in the presence of DNA damage. In eukaryotes, Rad6- and Rad18-mediated PCNA ubiquitination at lysine 164 promotes recruitment of TLS polymerases, allowing cells to efficiently cope with DNA damage. However, several studies showed that TLS polymerases can be recruited also in the absence of PCNA ubiquitination. We hypothesized that the stability of the interactions between DNA polymerase δ (Pol δ) subunits and/or between Pol δ and PCNA at the primer/template junction is a crucial factor to determine the requirement of PCNA ubiquitination. To test this hypothesis, we used a structural mutant of Pol δ in which the interaction between Pol3 and Pol31 is inhibited. We found that in yeast, rad18Δ-associated UV hypersensitivity is suppressed by pol3-ct, a mutant allele of the POL3 gene that encodes the catalytic subunit of replicative Pol δ. pol3-ct suppressor effect was specifically dependent on the Rev1 and Pol ζ TLS polymerases. This result strongly suggests that TLS polymerases could rely much less on PCNA ubiquitination when Pol δ interaction with PCNA is partially compromised by mutations. In agreement with this model, we found that the pol3-FI allele suppressed rad18Δ-associated UV sensitivity as observed for pol3-ct. This POL3 allele carries mutations within a putative PCNA Interacting Peptide (PIP) motif. We then provided molecular and genetic evidence that this motif could contribute to Pol δ-PCNA interaction indirectly, although it is not a bona fide PIP. Overall, our results suggest that the primary role of PCNA ubiquitination is to allow TLS polymerases to outcompete Pol δ for PCNA access upon DNA damage. Replicative DNA polymerases have the essential role of replicating genomic DNA during the S phase of each cell cycle. DNA replication occurs smoothly and accurately if the DNA to be replicated is undamaged. Conversely, replicative DNA polymerases stall abruptly when they encounter a damaged base on their template. In this case, alternative specialized DNA polymerases are recruited to insert nucleotides at sites of base lesions. However, these translesion polymerases are not processive and they are poorly accurate. Therefore, they need to be tightly regulated. This is achieved by the covalent binding of the small ubiquitin peptide to the polymerase cofactor PCNA that subsequently triggers the recruitment of translesion polymerases at sites of DNA damage. Yet, recruitment of translesion polymerases independently of PCNA ubiquitination also has been documented, although the underlying mechanism is not known. Moreover, this observation makes more difficult to understand the exact role of PCNA ubiquitination. Here, we present strong genetic evidence in Saccharomyces cerevisiae implying that the replicative DNA polymerase δ (Pol δ) prevents the recruitment of the translesion polymerases Pol ζ and Rev1 following UV irradiation unless PCNA is ubiquitinated. Thus, the primary role of PCNA ubiquitination would be to allow translesion polymerases to outcompete Pol δ upon DNA damage. In addition, our results led us to propose that translesion polymerases could be recruited independently of PCNA ubiquitination when Pol δ association with PCNA is challenged, for instance at difficult-to-replicate loci.
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影响因子：
3.8
作者：
Brocas, Clementine;Charbonnier, Jean-Baptiste;Maloisel, Laurent
通讯作者：
Maloisel, Laurent
影响因子：
7.7
作者：
Bomar, Martha G.;Pai, Ming-Tao;Zhou, Pei
通讯作者：
Zhou, Pei
影响因子：
16
作者：
Guo, Caixia;Sonoda, Eiichiro;Friedberg, Errol C.
通讯作者：
Friedberg, Errol C.
影响因子：
16
作者：
Haracska, L;Kondratick, CM;Prakash, L
通讯作者：
Prakash, L
DOI：
10.1073/pnas.0505949102
发表时间：
2005-12-20
影响因子：
11.1
作者：
Garg, P;Burgers, PM
通讯作者：
Burgers, PM