DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair.
The human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, we found that hRPA binds ssDNA with a defined polarity; a strong ssDNA interaction domain of hRPA is positioned at the 5' side of its binding region, a weak ssDNA-binding domain resides at the 3' side. Polarity appears crucial for positioning of the excision repair nucleases XPG and ERCC1-XPF on the DNA. With the 3'-oriented side of hRPA facing a duplex ssDNA junction, hRPA interacts with and stimulates ERCC1-XPF, whereas the 5'-oriented side of hRPA at a DNA junction allows stable binding of XPG to hRPA. Our data pinpoint hRPA to the undamaged strand during nucleotide excision repair. Polarity of hRPA on ssDNA is likely to contribute to the directionality of other hRPA-dependent processes as well.
Department of Cell Biology and Genetics, Medical Genetics Centre, Erasmus University, 3000 DR Rotterdam, The Netherlands.
W L
WL
de Laat
E
E
Appeldoorn
K
K
Sugasawa
E
E
Weterings
N G
NG
Jaspers
J H
JH
Hoeijmakers
D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism*; D006801:Humans; D007313:Insecta; D009687:Nuclear Proteins; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D051716:Replication Protein A; D013379:Substrate Specificity; D014157:Transcription Factors; D000099250:DNA Excision Repair Protein ERCC-5
D004247:DNA; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D004720:Endonucleases; D009687:Nuclear Proteins; D011506:Proteins; D051716:Replication Protein A; D014157:Transcription Factors; D000099250:DNA Excision Repair Protein ERCC-5; C497167:RPA1 protein, human; C071447:ERCC1 protein, human