2004 Fiscal Year Final Research Report Summary
Restoring DNA replication fork arrest within G-rich repetitive sequences by UP1 protein
| Project/Area Number |
15570131
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Functional biochemistry
|
|---|
| Research Institution | National Cancer Center (Research Institute) |
|---|
Principal Investigator |
FUKUDA Hirokazu National Cancer Center (Research Institute), Biochemistry Division, Section Head, 生化学部酵素化学研究室, 室長 (60300943)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
NAKAGAMA Hitoshi National Cancer Center (Research Institute), Biochemistry Division, Chief, 生化学部, 部長 (30198030)
|
|---|
| Project Period (FY) |
2003 – 2004
|
| Keywords | Repetive Sequences / Ouadruplex DNA / DNA Replication Fork / Minisatellite / Genomic Instability / Telomere / テロメア |
|---|
| Research Abstract |
DNA replication arrest at (CAGGG)n on a template plasmid could not been detected in an SV40 cell-free DNA replication system using extracts from 293 cells. It is possible that UP1 protein, a member of RecQ helicase family proteins, or any other factors in the extracts restored the replication arrest. It will be necessary to carry out similar experiments using extracts from the cells in which these factors are deficient or knocked down. There was no difference of the number of CGG repeat and chromosomal breaks between UP1-knocked-down HeLa cells and control cells. It is possible that leaky expression of UP1 or other UP1-like proteins compensated for the decrease in the amount of UP1 proteins. In order to clarify the functions of the hnRNP A3 protein, which was identified as another CAGGG repeat binding protein, we purified a recombinant protein from E.coli and examined the sequence-specificity of its DNA-binding. hnRNP A3 protein bound to d(CAGGG)n with a high affinity, and to telomeric d(TTAGGG)n repeats with a much higher affinity. In an in vitro DNA replication system using purified DNA polymerase and single-stranded DNA templates carrying (CAGGG)n or (TTAGGG)n repeats, hnRNP A3 proteins had an inhibitory effect on DNA synthesis within the repeats, contrary to UP1. hnRNP A3 protein was demonstrated in vitro to protect TTAGGG repeat from an attack of nucleases. We are now elucidating the effect of over-expression of hnRNP A3 on telomere-length in tet-on HeLa cells.
|
