The biophysical characterization of the interaction of XPA and the minimal DNA binding domain, XPA-MF122, with a mitomycin C-DNA interstrand crosslink

Despite the fact that numerous chemically and structurally diverse DNA lesions are repaired by nucleotide excision repair (NER), the mechanism of the interaction of XPA with damaged DNA is not well characterized. The overall goal of this study was the biophysical characterization of the interaction of XPA, with a mitomycin C (MMC)-NA interstrand crosslink. The 122 amino acid mini-domain (XPA-MF122) and the full-length XPA proteins were expressed, purified in high yield, and characterized by SDS-PAGE and Western analysis. Model 14- or 25-basepair MMC-interstrand crosslinked duplexes were synthesized, purified via chromatographic methods, and characterized by denaturing polyacrylamide gel electrophoresis, 32P-postlabeling, and circular dichroism (CD). The interaction of XPA-MF122 and XPA with the MMC-interstrand crosslinked duplexes was characterized through gel mobility shift assays and CD. In general, these proteins demonstrated a 2–3 fold greater affinity for MMC-crosslinked than unmodified duplexes. Despite this relatively modest preference for MMC-adducted DNA, XPA was observed to adopt a different conformation when bound to damaged compared to non-damaged DNA. Another aim of this study was to determine the effects of arsenic and cadmium on the ability of XPA to recognize and bind to MMC-DNA damage using CD and an optimized metal balanced gel shift assay. The changes in structure that were observed by CD for the Zn-XPA:MMC-DNA interaction were inhibited by cadmium substitution. In contrast, arsenic was shown to have little effect on XPA binding, even at high molar ratios of metal:protein. The final aim of this work was to characterize the response of XPA protein levels in human EJ-138 bladder cancer cells in response to the crosslinking agents MMC compared to UV. Western analysis of different cellular fractions in cells exposed to either UV or MMC appeared to cause a translocation of XPA from the cytosol to the nucleus. Defining the XPA:MMC-DNA interaction may aid in elucidating the mechanism by which DNA interstrand crosslinks and other forms of DNA damage are recognized and repaired by the NER machinery in eukaryotic cells. In addition, defining XPA-metal interactions may aid in understanding toxic metal suppression of DNA repair and the co-mutagenic and co-carcinogenic properties of these agents.