Abstract
Replication protein A (RPA), a heterotrimeric protein of 70-, 32-, and 14-kDa subunits, is an essential factor for DNA replication. Biochemical studies with human and yeast RPA have indicated that it is a DNA-binding protein that has higher affinity for single-stranded DNA. Interestingly, in vitro nucleotide excision repair studies with purified protein components have shown an absolute requirement for RPA in the incision of UV-damaged DNA. Here we use a mobility shift assay to demonstrate that human RPA binds a UV damaged duplex DNA fragment preferentially. Complex formation between RPA and the UV-irradiated DNA is not affected by prior enzymatic photo-reactivation of the DNA, suggesting an affinity of RPA for the (6-4) photoproduct. We also show that Mg2+ in the millimolar range is required for preferential binding of RPA to damaged DNA. These findings identify a novel property of RPA and implicate RPA in damage recognition during the incision of UV-damaged DNA.
Highlights
Nucleotide excision repair (NER)1 represents the most important cellular mechanism for repairing DNA damaged by ultraviolet (UV) light
replication protein A (RPA) purified is fully active in the in vitro replication of SV40 DNA [20], in nucleotide excision repair in a reconstituted system [7], and exhibits an affinity for ssDNA indistinguishable from that observed with RPA purified from HeLa cell extract [20]
We examined complex formation as a function of RPA concentration, using DNA fragment that had been irradiated with 15 kJ/m2 of UV
Summary
Nucleotide excision repair (NER) represents the most important cellular mechanism for repairing DNA damaged by ultraviolet (UV) light. Extensive genetic studies in Saccharomyces cerevisiae have indicated the requirement of the RAD1, RAD2, RAD3, RAD4, RAD10, RAD14, and RAD25 genes in the incision step of NER [1] In addition to their role in NER, RAD3 and RAD25 are essential for RNA polymerase II (Pol II) transcription [2,3,4], and their encoded proteins are constituents of Pol II transcription factor TFIIH [5, 6]. XPA binds damaged DNA, XPD and XPB are DNA helicases, and XPG and ERCC1-XPF encode the two nucleases (14 –18) In both the yeast and human systems, incision of UV-damaged DNA is absolutely dependent upon RPA [6, 7]. We show that RPA binds to UV-damaged DNA, identifying RPA as a damage recognition factor
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