Interactions of Human Nucleotide Excision Repair Protein XPA with DNA and RPA70ΔC327:  Chemical Shift Mapping and 15N NMR Relaxation Studies

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Amides, Chemical Structure, Genetics, Lesions, Peptides and Proteins

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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-rich C-terminal subdomain connected by a linker sequence. Here, the solution structure of XPA-MBD was further 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 a fragment of the largest subunit of replication protein A (RPA70ΔC327:  M1-Y326) was determined using chemical shift mapping. DNA binding in XPA-MBD was highly localized in the loop-rich subdomain for DNA with or without a lesion [dihydrothymidine (dhT) or 6−4-thymidine-cytidine (64TC)], or with DNA in single- or double-stranded form, indicating that the character of the lesion itself is not the driving force for 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 the loop-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 NMR relaxation data. The dynamic analyses for the XPA-MBD complexes with DNA revealed localized increases and decreases in S 2 and an increase in the global correlation time. Regions of XPA-MBD with the largest increases in S 2 overlapped regions having the largest chemical shifts changes upon binding DNA, indicating that the loop-rich subdomain becomes more rigid upon binding DNA. Interestingly, S 2 decreased for some residues in the zinc-binding core upon DNA association, indicating a possible concerted structural rearrangement on binding DNA.

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Biochemistry, v. 38, issue 46, p. 15116-15128