1999 Fiscal Year Final Research Report Summary
Replicational Errors Induced by Nucleoside Analogs in Cells
| Project/Area Number |
10044291
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| Research Category |
Grant-in-Aid for Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biological pharmacy
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| Research Institution | OKAYAMA UNIVERSITY |
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Principal Investigator |
HAYATSU Hikoya Okayama University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (10012593)
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| Co-Investigator(Kenkyū-buntansha) |
NEGISHI Kazuo Okayama University, Gene Research Center, Associate Professor, 遺伝子実験施設, 助教授 (70116490)
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| Project Period (FY) |
1998 – 1999
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| Keywords | Mismatch repair / Replicational errors / Retrovirus / MutS protein / Transition mutations / Error Catastrophe / mutL / Nucleoside analog |
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| Research Abstract |
Deoxyribosyl-dihydropyrimido[4,5-c] [1,2] oxazin-7-one (dP) is a potent mutagenic deoxycytidine analog capable of pairing with both A and G, thereby causing G-C to A-T and A-T to G-C transition mutations. We have found that the E. coli mismatch repair system can protect cells against this mutagenic action. dP is more mutagenic in mismatch-repair-defective mutH, mutL, and mutS strains than in a wild-type strain. At higher dP doses, the difference between the wild type and the mutator strains is smaller, suggesting that saturation of mismatch repair by dP may take place. Furthermore, introduction of a plasmid containing the mutLィイD1+ィエD1 gene into wile-type E. coli significantly reduced dP-induced mutagenesis. These results indicate that the mismatch repair system can remove replication errors induced by dP, but the capacity of this system to handle mismatches containing dP can be saturated. When cells were cultured at high dP concentration (20 μg/ml) final cell counts were reduced, and
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the frequency of RifィイD1rィエD1 mutants reached the high level of 10-ィイD14ィエD1. Colonies from those cultures were heterogeneously shaped, with 20-50% of them significantly smaller than control cells. We suggest that the cell killing and growth delay by dP are caused by excess mutations and saturation of mismatch repair (error catastrophe).
We also found that P-ribonucleoside triphosphate (Rptp) is efficiently incorporated into RNA transcripts in place of either UTP and/or CTP. The incorporation of such ambiguous analog into transcripts might specifically induce random mutations in retroviruses such as HIV, but not in the host cell genome. To test this we analyzed mutagenic effects of in an in vitro system mimicking a life cycle of retroviruses. The results showed that Rptp randomly accumulated mutations in the RNA transcripts during each cycle, and after 4 cycles of replication the mutation frequency was raised to 3.8%. Over 90% of mutations induced during the cycles were found to be C-to-U or U-to-C as expected. Less
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