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

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-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 (RPA70 Delta C327: 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. RPA70 Delta C327 was found to contact regions in both the zinc-binding and loop-rich subdomains. Some overlap of the DNA and RPA70 Delta C327 binding regions was observed in the loop-rich subdomain, indicating a possible cooperative DNA-binding mode between XPA and RPA70 Delta C327. 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 S2 and an increase in the global correlation time. Regions of XPA-MBD with the largest increases in S2 overlapped regions having the largest chemical shifts changes upon binding DNA, indicating that the loop-rich subdomain becomes more rigid upon binding DNA. Interestingly, S2 decreased for some residues in the zinc-binding core upon DNA association, indicating a possible concerted structural rearrangement on binding DNA.

Nucleotide Excision Repair by Mutant Xeroderma Pigmentosum Group A (XPA) Proteins with Deficiency in Interaction with RPA

“…However, the earlier finding, obtained by chemical shift mapping, which showed that RPA70(1-326) made contact with both the zinc-containing and C-terminal subdomains (41), is consistent with the results of this study. RPA70 and DNA were shown to share a binding surface in the C-terminal subdomain (41). Using direct cross-linking experiments, RPA70 and XPA were found to be positioned in close proximity to the damage site (42).…”

“…These results are not consistent with our previous study using RPA70(181-422) (34). However, the earlier finding, obtained by chemical shift mapping, which showed that RPA70(1-326) made contact with both the zinc-containing and C-terminal subdomains (41), is consistent with the results of this study. RPA70 and DNA were shown to share a binding surface in the C-terminal subdomain (41).…”

Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding

“…XPA is one of the damage recognition proteins involved in mammalian NER. The NMR structure of its DNA binding domain is known [49,50] and has recently been subjected to mutagenesis [51]. By monitoring luciferase expression following UV host cell reactivation in XPA deficient cells, the investigators assessed how various XPA DNA binding site mutants could initiate repair.…”

“…The "damaged" DNA oligonucleotide, F 26 50, consisted of a 50-mer with a centrally located fluorescein adduct thymine (FldT). The F 26 50 sequence was: 5′-GAC TAC GTA CTG TTA CGG CTC CAT C (FldT)C TAC CGC AAT CAG GCC AGA TCT GC-3′.…”

“…The complementary oligonucleotide, NDB, sequence contained a dA opposite the lesion. The damaged DNA, F 26 50, was 5′ end labeled using Optikinase (USB Corp.) and (γ-32 P) ATP (3000 Ci/mmol, GE Healthcare) according to standard procedures. The reactions were terminated by the addition of EDTA (20 mM) and the enzyme was heat denatured by incubation for 10 min at 65 °C.…”