Perrino Fred W, Blans Patrick, Harvey Scott, Gelhaus Stacy L, McGrath Colleen, Akman Steven A, Jenkins G Scott, LaCourse William R, Fishbein James C
Department of Biochemistry and Department of Cancer Biology, Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157, USA.
Chem Res Toxicol. 2003 Dec;16(12):1616-23. doi: 10.1021/tx034164f.
The effects of N(2)-ethylGua, O(6)-ethylGua, and O(6)-methylGua adducts in template DNA on polymerization by mammalian DNA polymerases alpha and eta have been investigated. The N(2)-ethylGua adduct blocks polymerization by the replicative DNA polymerase alpha to a much greater extent than does the O(6)-ethyl- or the O(6)-methylGua adducts. The DNA polymerase eta efficiently and accurately bypasses the N(2)-ethylGua lesion but like DNA polymerase alpha is similarly blocked by the O(6)-ethyl- or the O(6)-methylGua adducts. A steady state kinetic analysis of nucleotide insertion opposite the N(2)-ethylGua and the O(6)-ethylGua adducts by the DNA polymerases alpha and eta and extension from 3'-termini positioned opposite these adducts was performed to measure the efficiency and the accuracy of DNA synthesis past these lesions. This analysis showed that insertion of Cyt opposite the N(2)-ethylGua adduct by DNA polymerase alpha is approximately 10(4)-fold less efficient than insertion of Cyt opposite an unadducted Gua residue at the same position. Extension from the N(2)-ethylGua:Cyt 3'-terminus by DNA polymerase alpha is approximately 10(3)-fold less efficient than extension from a Cyt opposite the unadducted Gua. Insertion of Cyt opposite the N(2)-ethylGua lesion by the DNA polymerase eta is about 370-fold more efficient than by the DNA polymerase alpha, and extension from the N(2)-ethylGua:Cyt 3'-terminus by the DNA polymerase eta is about 3-fold more efficient than by the DNA polymerase alpha. Furthermore, the DNA polymerase eta preferably inserts the correct nucleotide Cyt opposite the N(2)-ethylGua lesion with nearly the same level of accuracy as opposite an unadducted Gua, thus minimizing the mutagentic potential of this lesion. This result contrasts with the relatively high misincorporation efficiency of Thy opposite the O(6)-ethylGua adduct by the DNA polymerases alpha and eta. In reactions containing both DNA polymerases alpha and eta, synthesis past the N(2)-ethylGua adduct is detected to permit completed replication of the adducted oligonucleotide template. These results suggest that accurate replication past the N(2)-ethylGua adduct might be facilitated in cells by pausing of replication catalyzed by DNA polymerase alpha and lesion bypass catalyzed by DNA polymerase eta.
研究了模板DNA中N(2)-乙基鸟嘌呤、O(6)-乙基鸟嘌呤和O(6)-甲基鸟嘌呤加合物对哺乳动物DNA聚合酶α和η聚合作用的影响。N(2)-乙基鸟嘌呤加合物比O(6)-乙基或O(6)-甲基鸟嘌呤加合物更能有效地阻断复制性DNA聚合酶α的聚合作用。DNA聚合酶η能高效且准确地绕过N(2)-乙基鸟嘌呤损伤，但与DNA聚合酶α一样，同样会被O(6)-乙基或O(6)-甲基鸟嘌呤加合物阻断。对DNA聚合酶α和η在N(2)-乙基鸟嘌呤和O(6)-乙基鸟嘌呤加合物相对位置处的核苷酸插入以及从与这些加合物相对的3'-末端延伸进行了稳态动力学分析，以测量DNA合成越过这些损伤的效率和准确性。该分析表明，DNA聚合酶α在N(2)-乙基鸟嘌呤加合物相对位置处插入胞嘧啶的效率比在相同位置未加合的鸟嘌呤残基处插入胞嘧啶的效率低约10(4)倍。DNA聚合酶α从N(2)-乙基鸟嘌呤:胞嘧啶3'-末端延伸的效率比从与未加合的鸟嘌呤相对的胞嘧啶延伸的效率低约10(3)倍。DNA聚合酶η在N(2)-乙基鸟嘌呤损伤相对位置处插入胞嘧啶的效率比DNA聚合酶α高约370倍，并且DNA聚合酶η从N(2)-乙基鸟嘌呤:胞嘧啶3'-末端延伸的效率比DNA聚合酶α高约3倍。此外，DNA聚合酶η更倾向于在N(2)-乙基鸟嘌呤损伤相对位置处插入正确的核苷酸胞嘧啶，其准确性与在未加合的鸟嘌呤相对位置处几乎相同，从而使该损伤的诱变潜力最小化。这一结果与DNA聚合酶α和η在O(6)-乙基鸟嘌呤加合物相对位置处错误掺入胸腺嘧啶的效率较高形成对比。在同时含有DNA聚合酶α和η的反应中，检测到越过N(2)-乙基鸟嘌呤加合物的合成，以允许加合的寡核苷酸模板完成复制。这些结果表明，在细胞中，DNA聚合酶α催化的复制暂停和DNA聚合酶η催化的损伤绕过可能有助于准确复制越过N(2)-乙基鸟嘌呤加合物。
