Plasmid P1 RepA is homologous to the F plasmid RepE class of initiators.
DNA replication of plasmid P1 requires a plasmid-encoded origin DNA-binding protein, RepA. RepA is an inactive dimer and is converted by molecular chaperones into an active monomer that binds RepA binding sites. Although the sequence of RepA is not homologous to that of F plasmid RepE, we found by using fold-recognition programs that RepA shares structural homology with RepE and built a model based on the RepE crystal structure. We constructed mutants in the two predicted DNA binding domains to test the model. As expected, the mutants were defective in P1 DNA binding. The model predicted that RepA binds the first half of the binding site through interactions with the C-terminal DNA binding domain and the second half through interactions with the N-terminal domain. The experiments supported the prediction. The model was further supported by the observation that mutants defective in dimerization map to the predicted subunit interface region, based on the crystal structure of pPS10 RepA, a RepE family member. These results suggest P1 RepA is structurally homologous to plasmid initiators, including those of F, R6K, pSC101, pCU1, pPS10, pFA3, pGSH500, Rts1, RepHI1B, RepFIB, and RSF1010.
Sharma
Suveena
S
Laboratory of Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Sathyanarayana
Bangalore K
BK
Bird
Jeremy G
JG
Hoskins
Joel R
JR
Lee
Byungkook
B
Wickner
Sue
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D029968:Escherichia coli Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry / Q000502:physiology*; D012097:Repressor Proteins / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011506:Proteins; C074164:RepE protein, E coli; D012097:Repressor Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004247:DNA; D004265:DNA Helicases