Binding of the human nucleotide excision repair proteins XPA and XPC/HR23B to the 5R-thymine glycol lesion and structure of the cis-(5R,6S) thymine glycol epimer in the 5′-GTgG-3′ sequence: destabilization of two base pairs at the lesion site

The 5R thymine glycol (5R-Tg) DNA lesion exists as a mixture of cis-(5R,6S) and trans-(5R,6R) epimers; these modulate base excision repair. We examine the 7:3 cis-(5R,6S):trans-(5R,6R) mixture of epimers paired opposite adenine in the 5′-GTgG-3′ sequence with regard to nucleotide excision repair. Human XPA recognizes the lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B recognition of Tg is superior. 5R-Tg is processed by the Escherichia coli UvrA and UvrABC proteins less efficiently than the C8-dG AAF adduct. For the cis-(5R, 6S) epimer Tg and A are inserted into the helix, remaining in the Watson–Crick alignment. The Tg N3H imine and A N6 amine protons undergo increased solvent exchange. Stacking between Tg and the 3′-neighbor G•C base pair is disrupted. The solvent accessible surface and T2 relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH3 group is favored; propeller twisting of the Tg•A pair and hydrogen bonding between Tg OH6 and the N7 atom of the 3′-neighbor guanine alleviate steric clash with the 5′-neighbor base pair. Tg also destabilizes the 5′-neighbor G•C base pair. This may facilitate flipping both base pairs from the helix, enabling XPC/HR23B recognition prior to recruitment of XPA

Redox-Dependent Formation of Disulfide Bonds in Human Replication Protein A

Human replication protein A (RPA) is a single-stranded DNA (ssDNA)-binding protein with three subunits. The largest subunit, p70, contains a conserved (cysteine)4-type zinc-finger motif that has been implicated in the regulation of DNA replication and repair. Previous studies indicated that the ssDNA-binding activity of RPA could be redox-regulated via reversible oxidation of cysteines in the zinc-finger motif. We exposed recombinant human RPA to hydrogen peroxide and characterized the oxidized protein by liquid chromatography/tandem mass spectrometric (LC/MS/MS) analyses. Our results demonstrated that, upon H2O2 treatment, four cysteines, which reside at the zinc-finger motif of the p70 subunit, could result in the formation of two pairs of intramolecular disulfides, Cys481-Cys486 and Cys500-Cys503; no cysteine sulfinic acid or cysteine sulfonic acid could be found. Moreover, the other 11 cysteines in this protein remained intact. The results demonstrated that the formation of disulfide bonds at the zinc-finger site was responsible for the redox regulation of the DNA-binding activity of RPA

Structural Characterization of Human RPA Sequential Binding to Single-Stranded DNA Using ssDNA as a Molecular Ruler

Human replication protein A (RPA), a heterotrimer composed of RPA70, RPA32, and RPA14 subunits, contains four single-stranded DNA (ssDNA) binding domains (DBD): DBD-A, DBD-B, and DBD-C in RPA70 and DBD-D in RPA32. Although crystallographic or NMR structures of these DBDs and a trimerization core have been determined, the structure of the full length of RPA or the RPA-ssDNA complex remains unknown. In this article, we have examined the structural features of RPA interaction with ssDNA by fluorescence spectroscopy. Using a set of oligonucleotides (dT) with varying lengths as a molecular ruler and also as the substrate, we have determined at single-nucleotide resolution the relative positions of the ssDNA with interacting intrinsic tryptophans of RPA. Our results revealed that Trp528 in DBD-C and Trp107 in DBD-D contact ssDNA at the 16th and 24th nucleotides (nt) from the 5′-end of the substrate, respectively. Evaluation of the relative spatial arrangement of RPA domains in the RPA-ssDNA complex suggested that DBD-B and DBD-C are spaced by about 4 nt (∼19 Å) apart, whereas DBD-C and DBD-D are spaced by about 7 nt (∼34 Å). On the basis of these geometric constraints, a global structure model for the binding of the major RPA DBDs to ssDNA was proposed

Structural Consequences of Epimerization of Thymine Glycol Lesions in Duplex DNA: Implications for DNA Repair

Thymine glycol (Tg), 5,6-dihydroxy-5,6-dihydrothymine, forms in DNA by reaction of thymine with reactive oxygen species. It exists as two diastereomeric pairs of epimers, the 5R cis, trans pair (5R,6S;5R,6R) and the 5S cis, trans pair (5S,6R; 5S,6S). The 5R pair is more abundant. At 30 °C, a 70%:30% cis:trans ratio of epimers is present in this sequence when 5R-Tg is opposite dA. For the cis epimer Tg and A remain in the Watson-Crick alignment. The Tg N3H imine and A N6 amine protons undergo increased solvent exchange. Stacking between Tg and the 3′-neighbor G·C base pair is disrupted. The solvent accessible surface and T2 relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH3 group is favored; propeller twisting of the Tg·A pair and hydrogen bonding between Tg OH6 and the N7 atom of the 3′-neighbor guanine alleviate steric clash with the 5′-neighbor base pair. Tg also destabilizes the 5′-neighbor G·C base pair. Under these conditions, the human NER protein XPA binds to the 5R-Tg lesion comparably to the C8-dG acetylaminoflourene (AAF) adduct, whereas XPC/HR23B binding of the Tg lesion. is superior than to the AAF adduct. In comparison, this lesion is processed by the Escherichia coli UvrA and UvrABC proteins less efficiently than the C8-dG AAF adduct. The destabilization of two base pairs by the cis epimer may facilitate flipping both base pairs from the helix, enabling XPC/HR23B binding prior to recruitment of XPA. When 5R-Tg pairs opposite dG in this sequence context only the cis epimer is observed. Tg assumes the wobble orientation and stacks below the 5′-neighbor dG, while the mismatched dG stacks below the 5′-neighbor dC. Stacking between Tg and the 3′-neighbor G·C base pair is disrupted. Differences in base excision repair of the Tg·G and Tg·A pairs by hNEIL1 may be related to the wobble orientation of the cis Tg epimer in the Tg·G pair, and the lack of hydrogen bonding between the Tg OH groups and the N7 atom of the 3′-neighbor dG. Hydrogen bonding between Tg6 OH6→G7 N7 in the Tg·A pair may increase the energetic barrier with regard to flipping of the Tg lesion into the active site pocket of the glycosylase, hindering repair

Specific and Efficient Binding of Xeroderma Pigmentosum Complementation Group A to Double-Strand/Single-Strand DNA Junctions With 3′- and/or 5′-SsDNA Branches

Human XPA is an important DNA damage recognition protein in nucleotide excision repair (NER). We previously observed that XPA binds to the DNA lesion as a homodimer [Liu, Y., Liu, Y., Yang, Z., Utzat, C., Wang, G., Basu, A. K., and Zou, Y. (2005) Biochemistry 44, 7361-7368]. Herein we report that XPA recognized undamaged DNA double-strand/single-strand (ds-ssDNA) junctions containing ssDNA branches with binding affinity (Kd = 49.1 ± 5.1 nM) much higher than its ability to bind to DNA damage. The recognized DNA junction structures include the Y-shape junction (with both 3′- and 5′-ssDNA branches), 3′-overhang junction (with a 3′-ssDNA branch), and 5′-overhang junction (with a 5′-ssDNA branch). Using gel filtration chromatography and gel mobility shift assays, we showed that the highly efficient binding appeared to be carried out by the XPA monomer and that the binding was largely independent of RPA. Furthermore, XPA efficiently bound to six-nucleotide mismatched DNA bubble substrates with or without DNA adducts including C8 guanine adducts of AF, AAF, and AP and the T[6,4]T photoproducts. Using a set of defined DNA substrates with varying degrees of DNA bending, we also found that the XPC-HR23B complex recognized DNA bending, whereas neither XPA nor the XPA-RPA complex could bind to bent DNA. We propose that, besides DNA damage recognition, XPA may also play a novel role in stabilizing, via its high affinity to ds-ssDNA junctions, the DNA strand opening surrounding the lesion for stable formation of preincision NER intermediates. Our results provide a plausible mechanistic interpretation for the indispensable requirement of XPA for both global genome and transcription-coupled repairs. Since ds-ssDNA junctions are common intermediates in many DNA metabolic pathways, the additional potential role of XPA in cellular processes is discussed. © 2006 American Chemical Society