Kinetic evidence for inefficient and error-prone bypass across bulky N2-guanine DNA adducts by human DNA polymerase ι
Kinetic evidence for inefficient and error-prone bypass across bulky N2-guanine DNA adducts by human DNA polymerase ι
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DOI：
10.1074/jbc.m600112200
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发表时间：
2006-05-05
影响因子：
4.8
通讯作者：
Guengerich, FP
中科院分区：
文献类型：
--
作者：
Choi, JY;Guengerich, FP
DNA聚合酶（pol）i已经被提出参与通过Hoogsteen碱基配对的小沟DNA加合物的跨损伤合成。G的N2位位于双链DNA的小沟侧，是各种致癌物修饰DNA的主要位点。分析在GN 2处具有不同加合物大小的寡核苷酸的旁路能力和与人聚合物的保真度。Poliota有效地绕过N-2-甲基（Me）G和N-2-乙基（Et）G，部分绕过N-2-异丁基（Ib）G和N-2-苄基G，并在N-2-CH 2（2-萘基）G（N-2-NaphG）、N-2-CH 2（9-蒽基）G（N-2-AnthG）和N-2-CH 2（6-苯并[a]芘基）G处被封闭。稳态动力学分析显示，根据大小，相对于N-2-G加合物，dCTP插入的k（cat）/K-m降低，相对于N-2-AnthG最大降低（61倍）。相对于模板G的dTTP错误插入频率增加3-11倍相对于加合物（N2-NaphG最高），表明体积（或可能的疏水性）对T错误掺入的累加效应。N-2-IbG、N-2-NaphG和N-2-AnthG与未修饰的G相比也降低了预稳态动力学爆发速率。与N-2-EtG和N-2-AnthG（但不是G）相反的高动力学硫代效应（Sp-2 '-脱氧胞苷5'-O-（1-硫代三磷酸））表明化学步骤在很大程度上受到加合物的干扰。与N-2-EtG相比，聚合对N-2，N-2-diMeG的严重抑制通过poleta而不是通过poliota与Hoogsteen碱基配对一致。因此，尽管Hoogsteen碱基配对的有利模式，但在G N2处的大体积基团严重抑制了通过poliota的聚合; poliota在细胞中大体积N-2-G加合物的跨损伤合成中可能发挥有限的作用。
DNA polymerase (pol) iota has been proposed to be involved in translesion synthesis past minor groove DNA adducts via Hoogsteen base pairing. The N2 position of G, located in minor groove side of duplex DNA, is a major site for DNA modification by various carcinogens. Oligonucleotides with varying adduct size at G N2 were analyzed for bypass ability and fidelity with human pol iota. Pol iota effectively bypassed N-2-methyl (Me) G and N-2-ethyl(Et)G, partially bypassed N-2-isobutyl(Ib)G and N-2-benzylG, and was blocked at N-2-CH2(2-naphthyl)G (N-2-NaphG), N-2-CH2(9-anthracenyl)G (N-2-AnthG), and N-2-CH2(6-benzo[a]pyrenyl)G. Steady-state kinetic analysis showed decreases of k(cat)/K-m for dCTP insertion opposite N-2-G adducts according to size, with a maximal decrease opposite N-2-AnthG (61-fold). dTTP misinsertion frequency opposite template G was increased 3-11-fold opposite adducts (highest with N2-NaphG), indicating the additive effect of bulk (or possibly hydrophobicity) on T misincorporation. N-2-IbG, N-2-NaphG, and N-2-AnthG also decreased the pre-steady-state kinetic burst rate compared with unmodified G. High kinetic thio effects (Sp-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) opposite N-2-EtG and N-2-AnthG (but not G) suggest that the chemistry step is largely interfered with by adducts. Severe inhibition of polymerization opposite N-2, N-2-diMeG compared with N-2-EtG by pol eta but not by pol iota is consistent with Hoogsteen base pairing by pol iota. Thus, polymerization by pol iota is severely inhibited by a bulky group at G N2 despite an advantageous mode of Hoogsteen base pairing; pol iota may play a limited role in translesion synthesis on bulky N-2-G adducts in cells.