Sloppy bypass of an abasic lesion catalyzed by a Y-family DNA polymerase
Sloppy bypass of an abasic lesion catalyzed by a Y-family DNA polymerase
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DOI：
10.1074/jbc.m610719200
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发表时间：
2007-03-16
影响因子：
4.8
通讯作者：
Suo, Zucai
中科院分区：
文献类型：
--
作者：
Fiala, Kevin A.;Suo, Zucai
逃避细胞修复途径的DNA损伤可以阻止复制机制并停止细胞周期。然而，这种损伤可以被Y家族DNA聚合酶绕过。在这里，DPO 4，一个典型的Y-家族成员从嗜热硫化叶菌solfataricus，被用来扩展我们的动力学研究的旁路的脱碱基位点，最诱变和无处不在的细胞病变之一。开发了一种短寡核苷酸测序测定法，以直接对Dpo 4合成的DNA旁路产物进行测序。我们的研究结果表明，掺入上游的无碱基病变是复制无差错的，但戏剧性的是，一旦DPO 4遇到病变，合成变得草率，旁路产品含有无数的诱变事件。在37 ° C下，与脱碱基病变相对的dAMP（29%）和dCMP（53%）的掺入与我们的动力学结果非常相关，并通过A规则和病变环出机制证明了两种主导的旁路途径。有趣的是，整体移码突变的百分比从71%（37摄氏度）增加到87%（75摄氏度）。进一步的分析表明，在较高温度下，通过A规则的病变旁路比病变环出机制强烈优选，并且伴随着减少在较低温度下观察到的与病变相对的“-1缺失”突变的发生。通过酶消化测定证实了通过后一种途径的旁路百分比，验证了我们的测序测定的可靠性。我们的研究结果表明，脱碱基病变导致DPO 4和可能的所有Y-家族成员从正常的复制模式切换到一个非常诱变的模式。
DNA damage that eludes cellular repair pathways can arrest the replication machinery and stall the cell cycle. However, this damage can be bypassed by the Y-family DNA polymerases. Here, Dpo4, an archetypal Y-family member from the thermophilic Sulfolobus solfataricus, was used to extend our kinetic studies of the bypass of an abasic site, one of the most mutagenic and ubiquitous cellular lesions. A short oligonucleotide sequencing assay is developed to directly sequence DNA bypass products synthesized by Dpo4. Our results show that incorporation upstream of the abasic lesion is replicated error-free; yet dramatically, once Dpo4 encounters the lesion, synthesis became sloppy, with bypass products containing a myriad of mutagenic events. Incorporation of dAMP (29%) and dCMP (53%) opposite the abasic lesion at 37 degrees C correlates exceptionally well with our kinetic results and demonstrates two dominant bypass pathways via the A-rule and the lesion loop-out mechanism. Interestingly, the percentage of overall frameshift mutations increased from 71 (37 degrees C) to 87% (75 degrees). Further analysis indicates that lesion bypass via the A-rule is strongly preferred over the lesion loop-out mechanism at higher temperatures and concomitantly reduces the occurrence of "-1 deletion" mutations observed opposite the lesion at lower temperatures. The bypass percentage via the latter pathway is confirmed by an enzymatic digestion assay, verifying the reliability of our sequencing assay. Our results demonstrate that an abasic lesion causes Dpo4 and possibly all Y-family members to switch from a normal to a very mutagenic mode of replication.