1996 Fiscal Year Final Research Report Summary
Mechanisims and Biological Significance of Recombination-dependent DNA Replication
| Project/Area Number |
07680675
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | The University of Tokyo |
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Principal Investigator |
MASAI Hisao University of Tokyo, Institute of Medical Science, Associate Professor, 医科学研究所, 助教授 (40229349)
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| Project Period (FY) |
1995 – 1996
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| Keywords | DNA Replication / DNA Recombination / Recombinational Repair / DNA Helicase / D-loop / R-loop / Adaptive Mutation / Double-stranded DNA Break |
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| Research Abstract |
(1) We have constructed PriA mutant genes containng point mutations in the ATP binding domain or in the cysteine residues of the putative Zinc-finger motif.
(2) Our preliminary results indicated essential roles of both ATP-binding and Zinc-finger motifs for stable DNA replication.
(3) PriA protein binds to the R-loop structure generated at the origin of ColE1-type plasmid in vitro.
(4) The N-terminal 300 amino acids are sufficient for binding to n'-pas, a hairpin which serves as an assembly site for the phiX174-type primosome.
(5) Serach of data base lead to identificatin of six bacterial genes structurally related to E.coli PriA.Interestingly, the putative Mycoplasma PriA homologue lacks a part of the ATP binding/helicase motif, although it contains the conserved zinc-finger motif.
(6) Search of the entire nucleotide sequence of the Saccharomyces cerevisiae genome did not result in identification of a gene with convincing homology to PriA.This indicates that the structure of an eukaryotic homologue of PriA,if any, may have diverged from bacterial PriA and its related genes.
(7) We have enzymatically characterized DnaA-dependent primosome assembled at the A site.
We have discovered a novel single-stranded DNA promoter recognized by RNA polymerase holoenzyme containing sigma^<70>. Termed RPO site, this sequence serves as a promoter for the genes downstream and also as the site for primer RNA synthesis for DNA replication. RPO site is an active promoter in the form of single-strand and in the presence of SSB.We have elucidated a mode of recognition of a unique secondary structure of RPO site by RNA polymerase.
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