Replication Protein A (RPA) is a single-stranded DNA binding protein that interacts with DNA repair proteins including Uracil DNA Glycosylase (UNG2). Here, I report DNA binding and activity assays using purified recombinant RPA and UNG2. Using synthetic DNA substrates, RPA was found to promote UNG2's interaction with ssDNA-dsDNA junctions regardless of the DNA strand polarity surrounding the junction. RPA stimulated UNG2's removal of uracil bases paired with adenine or guanine in DNA as much as 17-fold when the uracil was positioned 21 bps from ssDNA-dsDNA junctions, and the largest degree of UNG2 stimulation occurred when RPA was in molar excess compared to DNA. I found that RPA becomes sequestered on ssDNA regions surrounding junctions which promotes its spatial targeting of UNG2 near the junction. However, when RPA concentration exceeds free ssDNA, RPA promotes UNG2's activity without spatial constraints in dsDNA regions. These effects of RPA on UNG2 were found to be mediated primarily by interactions between RPA's winged-helix domain and UNG2's N-terminal domain, but when the winged-helix domain is unavailable, a secondary interaction between UNG2's N-terminal domain and RPA can occur. This work supports a widespread role for RPA in stimulating uracil base excision repair.

中文翻译：

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分析ssDNA-dsDNA连接处的复制蛋白A（RPA）对尿嘧啶DNA糖基化酶（UNG2）的刺激。

复制蛋白A（RPA）是一种单链DNA结合蛋白，可与包括尿嘧啶DNA糖基化酶（UNG2）在内的DNA修复蛋白相互作用。在这里，我报告使用纯化的重组RPA和UNG2进行DNA结合和活性测定。使用合成的DNA底物，发现RPA可以促进UNG2与ssDNA-dsDNA连接的相互作用，而不管连接周围的DNA链极性如何。当尿嘧啶距离ssDNA-dsDNA连接点21 bps时，RPA刺激UNG2对DNA中与腺嘌呤或鸟嘌呤配对的尿嘧啶碱基的去除高达17倍，而当RPA与DNA相比摩尔过量时，最大程度的UNG2刺激发生。我发现RPA被隔离在连接点周围的ssDNA区域上，这促进了它在连接点附近对UNG2的空间定位。然而，当RPA浓度超过游离ssDNA时，RPA会在dsDNA区域内不受空间限制的情况下促进UNG2的活性。发现RPA对UNG2的这些作用主要是由RPA的有翼螺旋结构域和UNG2的N末端结构域之间的相互作用介导的，但是当没有有翼的螺旋结构域时，UNG2的N末端结构域和RPA之间可能发生二次相互作用。 。这项工作支持RPA在刺激尿嘧啶基切除修复中的广泛作用。可能会出现N末端结构域和RPA。这项工作支持RPA在刺激尿嘧啶基切除修复中的广泛作用。可能会出现N末端结构域和RPA。这项工作支持RPA在刺激尿嘧啶基切除修复中的广泛作用。