Role for DNA polymerase κ in the processing of N2-N2-guanine interstrand cross-links
Role for DNA polymerase κ in the processing of N2-N2-guanine interstrand cross-links
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DOI：
10.1074/jbc.m801238200
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发表时间：
2008-06-20
影响因子：
4.8
通讯作者：
Lloyd, R. Stephen
中科院分区：
文献类型：
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作者：
Minko, Irina G.;Harbut, Michael B.;Lloyd, R. Stephen
虽然有令人信服的遗传证据同源重组独立的途径修复链间交联（ICLs），涉及translesion合成（TLS），这种模式缺乏生化支持。为了鉴定可能在TLS过去的ICL中起作用的DNA聚合酶，合成了含有位点特异性ICL的寡脱氧核苷酸，其中N-2-鸟嘌呤之间的连接类似于丝裂霉素C和烯醛形成的交联。在这里，数据显示，哺乳动物细胞复制的DNA含有这些病变是相似的97%的准确性。使用一系列的寡脱氧核苷酸，模拟潜在的中间体在ICL修复，我们证明，人类聚合酶（pol）κ不仅催化准确的掺入相反的交联鸟嘌呤，但也复制超出病变，从而提供了第一个生化证据TLS过去的ICL。通过截短非模板链的5'和3'末端，TLS的效率大大提高。进一步的分析表明，虽然酵母Rev 1可以结合一个dCTP对面的交联鸟嘌呤，没有证据表明TLS的pol zeta或pol zeta/Rev 1的组合。由于pol kappa能够绕过这些ICL，因此寻找pol kappa在耐受N-2-N-2-鸟嘌呤ICL中的作用的生物学证据;在暴露于丝裂霉素C后pol kappa耗尽的细胞中，细胞存活和染色体稳定性均受到不利影响。因此，生物化学数据和细胞研究都表明pol kappa在N-2-N-2-鸟嘌呤ICL的加工中的作用。
Although there exists compelling genetic evidence for a homologous recombination-independent pathway for repair of interstrand cross-links (ICLs) involving translesion synthesis (TLS), biochemical support for this model is lacking. To identify DNA polymerases that may function in TLS past ICLs, oligodeoxynucleotides were synthesized containing site-specific ICLs in which the linkage was between N-2-guanines, similar to cross-links formed by mitomycin C and enals. Here, data are presented that mammalian cell replication of DNAs containing these lesions was similar to 97% accurate. Using a series of oligodeoxynucleotides that mimic potential intermediates in ICL repair, we demonstrate that human polymerase (pol) kappa not only catalyzed accurate incorporation opposite the cross-linked guanine but also replicated beyond the lesion, thus providing the first biochemical evidence for TLS past an ICL. The efficiency of TLS was greatly enhanced by truncation of both the 5' and 3' ends of the nontemplating strand. Further analyses showed that although yeast Rev1 could incorporate a dCTP opposite the cross-linked guanine, no evidence was found for TLS by pol zeta or a pol zeta/Rev1 combination. Because pol kappa was able to bypass these ICLs, biological evidence for a role for pol kappa in tolerating the N-2-N-2-guanine ICLs was sought; both cell survival and chromosomal stability were adversely affected in pol kappa-depleted cells following mitomycin C exposure. Thus, biochemical data and cellular studies both suggest a role for pol kappa in the processing of N-2-N-2-guanine ICLs.