Distinct roles for Rev1p and Rev7p during translesion synthesis in Saccharomyces cerevisiae
Distinct roles for Rev1p and Rev7p during translesion synthesis in Saccharomyces cerevisiae
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DOI：
10.1046/j.1365-2958.1999.01583.x
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发表时间：
1999-10-01
影响因子：
3.6
通讯作者：
Fuchs, RPP
中科院分区：
文献类型：
--
作者：
Baynton, K;Bresson-Roy, A;Fuchs, RPP
酿酒酵母的翻译合成（TLS）至少需要Rev1p和聚合酶zeta (Pol zeta)，后者是Rev3聚合酶及其辅助因子Rev7p的复合体。尽管它们的确切作用尚不清楚，但体外研究表明，每种蛋白质都以依赖于特定病变和周围DNA序列的方式参与TLS。在本研究中，利用链分离分析试图确定Rev1和Rev7蛋白在TLS中的作用。该试验使用含有与复制阻断病变（n -2-乙酰氨基芴；AAF）相反的遗传标记的双链质粒，在体内定量和定性地测量TLS。AAF加合物以一种允许形成错位引物-模板复制中间体的方式定位在重复序列中。错位中间的延伸固定了移码突变（滑移TLS），而正确对齐的病变末端的延伸产生了无差错（非滑移TLS）。结果表明，在体内测量的Pol zeta介导的TLS中，Rev7p有很强的需求。此外，Rev1p只需要用于非滑动TLS；滑动TLS在没有它的情况下仍然有效，揭示了以前未被表征的Rev1p活性，类似于大肠杆菌的UmuDC功能。具体来说，这种活性对于从正确排列的病变末端延伸是必需的。
Translesion synthesis (TLS) in Saccharomyces cerevisiae requires at least Rev1p and polymerase zeta (Pol zeta), a complex of the Rev3 polymerase and its accessory factor Rev7p. Although their precise role(s) are poorly characterized, in vitro studies suggest that each protein contributes to TLS in a manner dependent on the particular lesion and surrounding DNA sequence. In the present study, strand segregation analysis is used to attempt to identify the role(s) of the Rev1 and Rev7 proteins during TLS. This assay uses double-stranded plasmids containing a genetic marker opposite to a replication blocking lesion (N-2-acetylaminofluorene; AAF) to measure TLS quantitatively and qualitatively in vivo. The AAF adduct is localized within a repetitive sequence in a manner that allows the formation of misaligned primer-template replication intermediates. Elongation from a misaligned intermediate fixes a frameshift mutation (slipped TLS), while extension of the correctly aligned lesion terminus yields error-free (non-slipped) TLS. The results indicate that there is a strong requirement for Rev7p during Pol zeta-mediated TLS measured in vivo. Furthermore, Rev1p is needed only for non-slipped TLS; slipped TLS remains efficient in its absence, revealing a previously uncharacterized Rev1p activity similar to Escherichia coli UmuDC function. Specifically, this activity is required for elongation from a correctly aligned lesion terminus.