Gourdin Audrey M, van Cuijk Loes, Tresini Maria, Luijsterburg Martijn S, Nigg Alex L, Giglia-Mari Guiseppina, Houtsmuller Adriaan B, Vermeulen Wim, Marteijn Jurgen A
Department of Genetics, Cancer Genomics Netherlands, Erasmus MC, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands.
Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 SM Amsterdam, The Netherlands; Swammerdam Institute for Life Sciences, University of Amsterdam, Sciencepark 904, 1098 XH Amsterdam, The Netherlands.
DNA Repair (Amst). 2014 Dec;24:46-56. doi: 10.1016/j.dnarep.2014.09.013. Epub 2014 Oct 16.
The ability of replication protein A (RPA) to bind single-stranded DNA (ssDNA) underlines its crucial roles during DNA replication and repair. A combination of immunofluorescence and live cell imaging of GFP-tagged RPA70 revealed that RPA, in contrast to other replication factors, does not cluster into replication foci, which is explained by its short residence time at ssDNA. In addition to replication, RPA also plays a crucial role in both the pre- and post-incision steps of nucleotide excision repair (NER). Pre-incision factors like XPC and TFIIH accumulate rapidly at locally induced UV-damage and remain visible up to 4h. However, RPA did not reach its maximum accumulation level until 3h after DNA damage infliction and a chromatin-bound pool remained detectable up to 8h, probably reflecting its role during the post-incision step of NER. During the pre-incision steps of NER, RPA could only be visualized at DNA lesions in incision deficient XP-F cells, however without a substantial increase in residence time at DNA damage. Together our data show that RPA is an intrinsically highly dynamic ssDNA-binding complex during both replication and distinct steps of NER.
复制蛋白A（RPA）结合单链DNA（ssDNA）的能力突显了其在DNA复制和修复过程中的关键作用。通过对绿色荧光蛋白标记的RPA70进行免疫荧光和活细胞成像相结合的研究发现，与其他复制因子不同，RPA不会聚集形成复制灶，这是由于其在ssDNA上的停留时间较短。除了复制，RPA在核苷酸切除修复（NER）的切口前和切口后步骤中也起着关键作用。像XPC和TFIIH这样的切口前因子会在局部诱导的紫外线损伤处迅速积累，并在长达4小时内保持可见。然而，RPA直到DNA损伤施加后3小时才达到其最大积累水平，并且与染色质结合的部分在长达8小时内仍可检测到，这可能反映了其在NER切口后步骤中的作用。在NER的切口前步骤中，只有在切口缺陷的XP-F细胞的DNA损伤处才能观察到RPA，然而其在DNA损伤处的停留时间并没有显著增加。我们的数据共同表明，在复制和NER的不同步骤中，RPA是一种本质上高度动态的ssDNA结合复合物。
