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首页> 外文期刊>Biochemistry >Characterization of Interstrand DNA-DNA Cross-Links Derived from Abasic Sites Using Bacteriophage phi 29 DNA Polymerase
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Characterization of Interstrand DNA-DNA Cross-Links Derived from Abasic Sites Using Bacteriophage phi 29 DNA Polymerase

机译：使用噬菌体phi 29 DNA聚合酶表征无碱基位点的链间DNA-DNA交联。

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Interstrand cross-links in cellular DNA are highly deleterious lesions that block transcription and replication. We recently characterized two new structural types of interstrand cross-links derived from the reaction of abasic (Ap) sites with either guanine or adenine residues in duplex DNA. Interestingly, these Ap-derived cross-links are forged by chemically reversible processes, in which the two strands of the duplex are joined by hemiaminal, imine, or aminoglycoside linkages. Therefore, understanding the stability of Ap-derived cross-links may be critical in defining the potential biological consequences of these lesions. Here we employed bacteriophage phi 29 DNA polymerase, which can couple DNA synthesis and strand displacement, as a model system to examine whether dA-Ap cross-links can withstand DNA-processing enzymes. We first demonstrated that a chemically stable interstrand cross-link generated by hydride reduction of the dG-Ap cross-link completely blocked primer extension by phi 29 DNA polymerase at the last unmodified nudeobase preceding cross-link. We then showed that the nominally reversible dA-Ap cross-link behaved, for all practical purposes, like an irreversible, covalent DNA DNA cross-link. The dA-Ap cross-link completely blocked progress of the phi 29 DNA polyrnerase at the last unmodified base before the cross-link. This suggests that Ap-derived cross-links have the power to block various DNA-processing enzymes in the cell. In addition, our results reveal phi 29 DNA polymerase as a tool for detecting the presence and mapping the location of interstrand cross-links (and possibly other lesions) embedded within regions of duplex DNA.
机译：细胞DNA中的链间交联是高度有害的损伤，会阻止转录和复制。我们最近表征了两个新的结构类型的链间交联，其源自双碱基DNA中无碱基（Ap）位点与鸟嘌呤或腺嘌呤残基的反应。有趣的是，这些Ap衍生的交联是通过化学可逆过程锻造的，其中双链体的两条链通过半胱氨酸，亚胺或氨基糖苷键连接。因此，了解Ap衍生的交联的稳定性对于定义这些病变的潜在生物学后果可能至关重要。在这里，我们采用了可以结合DNA合成和链置换的噬菌体phi 29 DNA聚合酶作为模型系统，以检查dA-Ap交联是否可以承受DNA加工酶。我们首先证明，由dG-Ap交联物的氢化物还原产生的化学稳定的链间交联完全阻断了phi 29 DNA聚合酶在交联之前的最后一个未修饰的裸碱基上的引物延伸。然后，我们表明，出于所有实际目的，名义上可逆的dA-Ap交联表现为不可逆的共价DNA DNA交联。 dA-Ap交联完全阻止了phi 29 DNA聚合酶在交联前的最后一个未修饰碱基处的进展。这表明Ap衍生的交联具有阻断细胞中各种DNA处理酶的能力。此外，我们的研究结果揭示了phi 29 DNA聚合酶是一种检测双链DNA区域内嵌入的链间交联（以及可能存在其他病变）的位置并作图的工具。

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