1991 Fiscal Year Final Research Report Summary
Involvement of Chromosome in Regulation of DNA Replication and Repair
| Project/Area Number |
02454489
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | RIKEN (The Institute of Physical and Chemical Research) |
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Principal Investigator |
HANAOKA Fumio RIKEN, Cell. Physiol. Lab., Chief Scientist, 細胞生理学研究室, 主任研究員 (50012670)
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| Co-Investigator(Kenkyū-buntansha) |
SUGASAWA Kaoru RIKEN, Cell. Physiol. Lab., Research Scientist, 細胞生理学研究室, 研究員 (70202124)
MIYAZAWA Hiroshi RIKEN, Cell. Physiol. Lab., Research Scientist, 細胞生理学研究室, 研究員 (40183967)
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| Project Period (FY) |
1990 – 1991
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| Keywords | SV40 virus / minichromosome / cell-free replication system / nucleosome / DNA topoisomerase / UV light / cell-free repair system / xeroderma pigmentosum |
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| Research Abstract |
We have developed a call-free system for replication of SV40 minichromosomes with purified proteins.
Since this reconstituted system lacked activity of de novo nucleosome assembly, we could specifically examine the behavior of parental histones during chromosome replication. Solution hybridization analyses suggested that the histones were not completely detached from but somehow associated with template DNA. Using strand-specific probes and MNase digested nascent mononucleosomal DNA, we obtained the results indicating that the parental histone octamers were segregated distributively between leading and lagging strands during the chromosome replication with purified proteifis.
When SV40 chromosomes were replicated with topoI alone as a swivelase, the reaction produced shorter leading strands but those of mature size were accumulated in the reaction supplemented with topo II. These results indicate that topo II has a crucial role as a swivelase in the late stage of SV40 chromosome replicat
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ion in vitro.
On the other hand, we have developed a cell-free system supporting repair reactions on DNA organized into hucleosomes, that is, SV40 chromosomes. Standard reaction mixtures contained UV-irradiated SV40 chromosomes, unirradiated plasmid DNA, whole cell extracts of HeLa cells(Manley's extracts), ATP-regenerating system and several low molecular weight materials including[alpha-^<32>P]dCTP. After incubated at 30ﾟC, DNAs were isolated, digested with EcoRI and separated by agarose gel electrophoresis. Repair synthesis as well as nick-translation of DNAs were detected by autoradiography. DNA synthesis on SV40 DNA was dependent on doses of irradiated DNA. When we used cell extracts made from XP-A or XP-C cells in place of HeLa cell extracts, the UV-dependent DNA synthesis decreased remarkably. Mixing of XP-A and XP-
C cell extracts restored the DNA synthesis to similar level as HeLa cell extracts. Thus our in vitro system appears to be useful for reconstituting at least some features of chromosomal DNA excision repair. Less
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