DNA damaging agents have been shown to stimulate the localization of human DNA helicase B (HDHB) in nuclear foci, suggesting that HDHB might participate in DNA damage response. In the first part of this dissertation, we found that the chromatin-associated fraction of HDHB increases in cells exposed to a variety of DNA damaging agents. HDHB chromatin accumulation is most prominent in S phase cells exposed to agents that cause replication fork stalling or collapse. Inhibition of checkpoint kinases does not prevent damage-induced accumulation of HDHB on chromatin, suggesting that HDHB associates directly with DNA lesions or with other proteins recruited to lesions. Silencing of HDHB does not affect UV-induced RPA focus formation, but diminishes induction of TopBP1 foci. DNA damage induced CHK1 phosphorylation is impaired in HDHB-depleted cells. These results identify HDHB as a novel factor that associates with damaged chromatin and promotes intra-S phase damage responses.
In the second part of this dissertation, we further investigated the possible function of HDHB in homologous recombination. HDHB-depleted cells showed more aphidicolin-induced chromosome breaks than control-depleted cells. HDHB-depleted cells have fewer sister chromatid exchange than control-depleted cells. An in vivo recombination assay showed that HDHB silencing results in impaired homologous recombination. In vitro, recombinant HDHB stimulates Rad51-mediated 5-3 heteroduplex extension. Our studies suggest a function of HDHB in stimulating ssDNA/duplex structure during homologous recombination.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-09152007-110210 |
| Date | 03 October 2007 |
| Creators | Liu, Hanjian |
| Contributors | Eugene Oltz, David Cortez, Ellen Fanning, James Patton, Jennifer Pietenpol |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu//available/etd-09152007-110210/ |
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