Replication Protein A Prohibits Diffusion of the PCNA Sliding Clamp along Single-Stranded DNA.
Replication Protein A Prohibits Diffusion of the PCNA Sliding Clamp along Single-Stranded DNA.
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DOI：
10.1021/acs.biochem.6b01213
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发表时间：
2017-04-04
期刊：
影响因子：
2.9
通讯作者：
Benkovic SJ
中科院分区：
文献类型：
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作者：
Hedglin M;Benkovic SJ
复制型聚合酶不能适应天然DNA序列的畸变，例如暴露于反应性代谢物和环境诱变剂对天然模板碱基的修饰（损伤）。因此，当遇到这些损伤时，受影响的模板上的DNA合成突然停止，但复制叉继续前进，在最终停止之前暴露出长时间的受损模板。这种停滞可以通过跨损伤DNA合成（TLS）来克服，其中专门的TLS聚合酶与驻留的增殖细胞核抗原（PCNA）结合并复制受损的DNA。因此，TLS的一个关键方面是在通过复制聚合酶从DNA合成中解偶联叉进展时将PCNA保持在封闭的引物/模板（P/T）接合处或附近。单链DNA（ssDNA）结合蛋白，复制蛋白A（RPA），覆盖暴露的模板，并可能阻止PCNA沿着邻近封闭的P/T连接的单链DNA扩散。然而，这个想法还没有得到直接的验证。我们最近开发了一种独特的Cy 3-Cy 5 Forster共振能量转移（FRET）对，直接报告PCNA对DNA的占有率。在这项研究中，我们利用这种FRET对直接和连续监测的保留人PCNA在一个封闭的P/T连接。从广泛的稳态和预稳态FRET测定的结果表明，RPA紧密结合的ssDNA附近的一个封闭的P/T交界处，并限制PCNA的上游双链体区域的物理阻断扩散的PCNA沿着ssDNA。
The replicative polymerases cannot accommodate distortions to the native DNA sequence such as modifications (lesions) to the native template bases from exposure to reactive metabolites and environmental mutagens. Consequently, DNA synthesis on an afflicted template abruptly stops upon encountering these lesions, but the replication fork progresses onward, exposing long stretches of the damaged template before eventually stalling. Such arrests may be overcome by translesion DNA synthesis (TLS) in which specialized TLS polymerases bind to the resident proliferating cell nuclear antigen (PCNA) and replicate the damaged DNA. Hence, a critical aspect of TLS is maintaining PCNA at or near a blocked primer/template (P/T) junction upon uncoupling of fork progression from DNA synthesis by the replicative polymerases. The single-stranded DNA (ssDNA) binding protein, replication protein A (RPA), coats the exposed template and might prohibit diffusion of PCNA along the single-stranded DNA adjacent to a blocked P/T junction. However, this idea had yet to be directly tested. We recently developed a unique Cy3-Cy5 Forster resonance energy transfer (FRET) pair that directly reports on the occupancy of DNA by PCNA. In this study, we utilized this FRET pair to directly and continuously monitor the retention of human PCNA at a blocked P/T junction. Results from extensive steady state and pre-steady state FRET assays indicate that RPA binds tightly to the ssDNA adjacent to a blocked P/T junction and restricts PCNA to the upstream duplex region by physically blocking diffusion of PCNA along ssDNA.