Recognition of cisplatin-DNA interstrand cross-links by replication protein A.
Replication protein A (RPA) is a heterotrimeric protein that is required for DNA replication and most DNA repair pathways. RPA has previously been shown to play a role in recognizing and binding damaged DNA during nucleotide excision repair (NER). RPA has also been suggested to play a role in psoralen DNA interstrand cross-link (ICL) repair, but a clear biochemical activity has yet to be identified in the ICL DNA repair pathways. Using HeLa cell extracts and DNA affinity chromatography, we demonstrate that RPA is preferentially retained on a cisplatin interstrand cross-link (ICL) DNA column compared with undamaged DNA. The retention of RPA on cisplatin intrastrand and ICL containing DNA affinity columns is comparable. In vitro electrophoretic mobility shift assays (EMSAs) using synthetic DNA substrates and purified RPA demonstrate higher affinity for cisplatin ICL DNA binding compared with undamaged DNA. The enhanced binding of RPA to the cisplatin ICL is dependent on the DNA length. As the DNA flanking the cisplatin ICL is increased from 7 to 21 bases, preferential RPA binding is observed. Fluorescence anisotropy reveals greater than 200-fold higher affinity to a cisplatin ICL containing 42-mer DNA compared with an undamaged DNA and a 3-4-fold higher affinity when compared with a cisplatin intrastrand damaged DNA. As the DNA length and stringency of the binding reaction increase, greater preferential binding of RPA to cisplatin ICL DNA is observed. These data are consistent with a role for RPA in the initial recognition and initiation of cisplatin ICL DNA repair.
Patrick
Steve M
SM
Department of Biochemistry and Cancer Biology, University of Toledo Health Science Campus, Toledo, Ohio 43614, USA. Stephan.Patrick@utoledo.edu
Tillison
Kristin
K
Horn
Jeffrey M
JM
D002945:Cisplatin / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry* / Q000378:metabolism; D005454:Fluorescence Polarization; D006801:Humans; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000378:metabolism; D051716:Replication Protein A / Q000737:chemistry / Q000378:metabolism*
D003432:Cross-Linking Reagents; D018736:DNA Adducts; D009692:Nucleic Acid Heteroduplexes; D051716:Replication Protein A; D004247:DNA; D002945:Cisplatin