title: An affinity of human replication protein A for ultraviolet-damaged DNA.
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.
authors.affiliation: Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555-1061, USA.
authors.forename: J L
authors.initials: JL
authors.lastname: Burns
authors.forename: S N
authors.initials: SN
authors.lastname: Guzder
authors.forename: P
authors.initials: P
authors.lastname: Sung
authors.forename: S
authors.initials: S
authors.lastname: Prakash
authors.forename: L
authors.initials: L
authors.lastname: Prakash
mesh_terms: D004247:DNA / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D051716:Replication Protein A; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays
chemical_list: D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D004247:DNA