Title

Structural and functional studies of the human DNA repair protein XRCC1

Date of Completion

January 2001

Keywords

Biology, Molecular

Degree

Ph.D.

Abstract

XRCC1 (X-ray cross-complementing group 1) is an essential protein required for single-strand DNA break repair. An XRCC1 knockout is embryonic-lethal in mice and cells deficient in XRCC1 have increased sensitivity to X-ray, UV, ethylmethane sulfonate and other DNA damaging agents. ^ XRCC1 plays important roles in the detection and repair of single-strand DNA breaks: it promotes the assembly of the DNA repair complexes through interactions with DNA polymerase β (β-Pol), poly-(ADP-ribose) polymerase (PARP), DNA lipase III, polynuclcetide kinase/phosphatase (PNK), and DNA; XRCC1 may also stimulate the activity of some of the repair enzymes; and XRCC1 helps protect DNA in the vicinity of the single-strand break from further damage. ^ The main objective of this thesis research was to characterize in detail the structural basis for the interaction of XRCC1 with β-Pol and the formation of the single-strand DNA break repair complexes. The NMR solution structure of the β-Pol-binding N-terminal domain of XRCC1 (XRCC1-NTD) was determined. Using biochemical and genetical approaches, the interaction between XRCC1 and β-Pol was localized to the C-terminal thumb domain of β-Pol and XRCC1-NTD. The interacting surfaces of both proteins were identified by NMR chemical shift mapping and site-directed mutagenesis. The XRCC1-NTD was also found to bind DNA. A structural model for the interaction of XRCC1-NTD with β-Pol and gapped DNA was proposed and experimentally tested. According to this model, XRCC1-NTD and β-Pol binding together to gapped DNA surround the DNA break and protect it from possible further damage. The XRCC1-NTD binds near the active site of β-Pol, which supports a possible role for XRCC1 in regulation of β-Pol activity. The domain structure of XRCC1 was mapped and two new potentially important regions were identified. One of them, the inner domain (ID), was found to be involved in DNA binding, bringing the number of DNA binding domains of XRCC1 to three, two of which were identified in this work. ^ Our results emphasize the central role XRCC1 plays in single-strand DNA break repair and give rise to new ideas for the mechanisms of assembly, function and regulation the single-strand break DNA repair complexes. ^