Yeast DNA polymerase ζ maintains consistent activity and mutagenicity across a wide range of physiological dNTP concentrations.
Yeast DNA polymerase ζ maintains consistent activity and mutagenicity across a wide range of physiological dNTP concentrations.
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DOI：
10.1093/nar/gkw1149
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发表时间：
2017-02-17
影响因子：
14.9
通讯作者：
Shcherbakova PV
中科院分区：
文献类型：
--
作者：
Kochenova OV;Bezalel-Buch R;Tran P;Makarova AV;Chabes A;Burgers PM;Shcherbakova PV
在酵母中，dNTP池在DNA损伤反应期间急剧扩大。我们发现在菌株中也存在类似的dNTP升高，在这些菌株中，固有的复制体缺陷促进了容易出错的DNA聚合酶ζ(POLζ)参与未损伤DNA的复制。为了了解dNTP池增加对Polζ功能的意义，我们研究了四亚单位Polζ(Polζ4)和Polζ4-Rev1(Polζ5)复合体在“正常S时相”和“损伤-反应”dNTP浓度下的活性和保真度。REV1的存在抑制了POLζ的活性，并极大地增加了Polζ4单独造成的三个‘X-dCTP’错配率。Polζ4和Polζ5在G核苷酸上的错配程度最高，并频繁地产生多个紧密间隔的序列变化。令人惊讶的是，从‘S期’到‘损伤-反应’dNTP水平的变化对POLζ复合体的活性、保真度和错误特异性影响很小。此外，当羟基脲抑制dNTP的合成时，体内由复制体缺陷或紫外线辐射引发的依赖POLζ的突变并未减少，表明POLζ的功能不需要高水平的dNTP。结果支持这样一个模型，即需要dNTP的上调来促进非诱变耐受途径，而POLζ合成代表了当dNTP供应较少时拯救停滞复制的独特机制。
In yeast, dNTP pools expand drastically during DNA damage response. We show that similar dNTP elevation occurs in strains, in which intrinsic replisome defects promote the participation of error-prone DNA polymerase ζ (Polζ) in replication of undamaged DNA. To understand the significance of dNTP pools increase for Polζ function, we studied the activity and fidelity of four-subunit Polζ (Polζ4) and Polζ4-Rev1 (Polζ5) complexes in vitro at ‘normal S-phase’ and ‘damage-response’ dNTP concentrations. The presence of Rev1 inhibited the activity of Polζ and greatly increased the rate of all three ‘X-dCTP’ mispairs, which Polζ4 alone made extremely inefficiently. Both Polζ4 and Polζ5 were most promiscuous at G nucleotides and frequently generated multiple closely spaced sequence changes. Surprisingly, the shift from ‘S-phase’ to ‘damage-response’ dNTP levels only minimally affected the activity, fidelity and error specificity of Polζ complexes. Moreover, Polζ-dependent mutagenesis triggered by replisome defects or UV irradiation in vivo was not decreased when dNTP synthesis was suppressed by hydroxyurea, indicating that Polζ function does not require high dNTP levels. The results support a model wherein dNTP elevation is needed to facilitate non-mutagenic tolerance pathways, while Polζ synthesis represents a unique mechanism of rescuing stalled replication when dNTP supply is low.