Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins.
Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins.
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DOI：
10.1093/nar/gku1321
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发表时间：
2015-01
影响因子：
14.9
通讯作者：
Doherty AJ
中科院分区：
文献类型：
--
作者：
Guilliam TA;Jozwiakowski SK;Ehlinger A;Barnes RP;Rudd SG;Bailey LJ;Skehel JM;Eckert KA;Chazin WJ;Doherty AJ
PrimPol是最近鉴定的参与真核DNA损伤耐受的聚合酶，用于再引发和跨损伤合成机制以绕过核和线粒体DNA损伤。在这份报告中，我们研究了如何调节人类PrimPol的酶活性。我们发现，与其他TLS聚合酶不同，PrimPol不受PCNA刺激，也不与其在体内相互作用。我们确定PrimPol在体内与两种主要的单链结合蛋白RPA和mtSSB相互作用。使用NMR光谱，我们表征了负责PrimPol-RPA相互作用的结构域，揭示了PrimPol直接与RPA 70的N-末端结构域结合。与SSB在刺激复制聚合酶中的既定作用相反，我们发现SSB显著限制PrimPol的引发酶和聚合酶活性。为了确定这种调节的要求，我们采用了两种正向突变测定来表征PrimPol的复制保真度。我们发现PrimPol是一种诱变聚合酶，具有独特的错误特异性，高度偏向于插入-缺失错误。鉴于PrimPol的易错性，我们提出了一种机制，即SSBs极大地限制了这种酶在停滞叉处对DNA复制的贡献，从而降低了PrimPol在基因组复制过程中的致突变潜力。
PrimPol is a recently identified polymerase involved in eukaryotic DNA damage tolerance, employed in both re-priming and translesion synthesis mechanisms to bypass nuclear and mitochondrial DNA lesions. In this report, we investigate how the enzymatic activities of human PrimPol are regulated. We show that, unlike other TLS polymerases, PrimPol is not stimulated by PCNA and does not interact with it in vivo. We identify that PrimPol interacts with both of the major single-strand binding proteins, RPA and mtSSB in vivo. Using NMR spectroscopy, we characterize the domains responsible for the PrimPol-RPA interaction, revealing that PrimPol binds directly to the N-terminal domain of RPA70. In contrast to the established role of SSBs in stimulating replicative polymerases, we find that SSBs significantly limit the primase and polymerase activities of PrimPol. To identify the requirement for this regulation, we employed two forward mutation assays to characterize PrimPol's replication fidelity. We find that PrimPol is a mutagenic polymerase, with a unique error specificity that is highly biased towards insertion-deletion errors. Given the error-prone disposition of PrimPol, we propose a mechanism whereby SSBs greatly restrict the contribution of this enzyme to DNA replication at stalled forks, thus reducing the mutagenic potential of PrimPol during genome replication.
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影响因子：
16
作者：
Bianchi, Julie;Rudd, Sean G.;Jozwiakowski, Stanislaw K.;Bailey, Laura J.;Soura, Violetta;Taylor, Elaine;Stevanovic, Irena;Green, Andrew J.;Stracker, Travis H.;Lindsay, Howard D.;Doherty, Aidan J.
通讯作者：
Doherty, Aidan J.
影响因子：
3.8
作者：
Lehmann, Alan R.;Niimi, Atsuko;Green, Catherine M.
通讯作者：
Green, Catherine M.
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
影响因子：
14.9
作者：
Furukohri, Asako;Nishikawa, Yoshito;Maki, Hisaji
通讯作者：
Maki, Hisaji
影响因子：
14.9
作者：
Jozwiakowski SK;Keith BJ;Gilroy L;Doherty AJ;Connolly BA
通讯作者：
Connolly BA