The N-terminal domain of the large subunit of human replication protein A binds to Werner syndrome protein and stimulates helicase activity.
Werner syndrome (WS) is a recessive inherited human disease characterized by the early onset of aging. The gene mutated in WS encodes a DNA helicase that unwinds the double helical structure of DNA in the 3'-->5' direction as well as a 3'-->5' exonuclease. Our previous studies indicated that the activity of Werner syndrome helicase (WRN) could be stimulated by human replication protein A (hRPA), a heterotrimeric single-stranded DNA binding protein. We now localize the interaction between WRN and hRPA by measuring the stimulation of helicase activity and the binding of WRN by hRPA and its derivatives. The large subunit of hRPA (hRPA70) stimulates WRN helicase to the same extent as the hRPA heterotrimer, whereas the dimer of the two smaller subunits (hRPA 32.14) does not stimulate. By examining hRPA70 mutants with progressive deletions from either the C- or N-terminus, we found that the domain responsible for stimulation lies in the N-terminal half of the protein. By using enzyme-linked immunosorbent assay (ELISA) to examine physical interaction between WRN and the same deletion mutants, we found that the WRN-binding motif is located within amino acids 100-300 and overlaps with the single-stranded DNA binding domain (amino acids 150-450). We suggest that hRPA, by engaging in both protein-protein and protein-DNA interactions, facilitates unwinding events catalyzed by WRN helicase during DNA synthetic processes. These data should help further elucidation of the molecular mechanisms of genetic instability and premature aging phenotypes manifested by WS.
D000595:Amino Acid Sequence; D004265:DNA Helicases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005090:Exodeoxyribonucleases; D006801:Humans; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D053484:RecQ Helicases; D051716:Replication Protein A; D014898:Werner Syndrome / Q000378:metabolism*; D000071657:Werner Syndrome Helicase
D004268:DNA-Binding Proteins; D020033:Protein Isoforms; C497167:RPA1 protein, human; D051716:Replication Protein A; D005090:Exodeoxyribonucleases; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase