| Abstract
| - Human XPA is an essential component in the multienzyme nucleotide excision repair (NER)pathway. The solution structure of the minimal DNA binding domain of XPA (XPA-MBD: M98-F219)was recently determined [Buchko et al. (1998) Nucleic Acids Res. 26, 2779−2788, Ikegami et al. (1998)Nat. Struct. Biol. 5, 701−706] and shown to consist of a compact zinc-binding core and a loop-richC-terminal subdomain connected by a linker sequence. Here, the solution structure of XPA-MBD wasfurther refined using an entirely new class of restraints based on pseudocontact shifts measured in cobalt-substituted XPA-MBD. Using this structure, the surface of XPA-MBD which interacts with DNA and afragment of the largest subunit of replication protein A (RPA70ΔC327: M1-Y326) was determined usingchemical shift mapping. DNA binding in XPA-MBD was highly localized in the loop-rich subdomain forDNA with or without a lesion [dihydrothymidine (dhT) or 6−4-thymidine-cytidine (64TC)], or with DNAin single- or double-stranded form, indicating that the character of the lesion itself is not the driving forcefor XPA binding DNA. RPA70ΔC327 was found to contact regions in both the zinc-binding and loop-rich subdomains. Some overlap of the DNA and RPA70ΔC327 binding regions was observed in theloop-rich subdomain, indicating a possible cooperative DNA-binding mode between XPA and RPA70ΔC327.To complement the chemical shift mapping data, the backbone dynamics of free XPA-MBD and XPA-MBD bound to DNA oligomers containing dhT or 64TC lesions were investigated using 15N NMRrelaxation data. The dynamic analyses for the XPA-MBD complexes with DNA revealed localized increasesand decreases in S 2 and an increase in the global correlation time. Regions of XPA-MBD with the largestincreases in S 2 overlapped regions having the largest chemical shifts changes upon binding DNA, indicatingthat the loop-rich subdomain becomes more rigid upon binding DNA. Interestingly, S 2 decreased forsome residues in the zinc-binding core upon DNA association, indicating a possible concerted structuralrearrangement on binding DNA.
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