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

Abstract

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 N(6) amine protons undergo increased solvent exchange. Stacking between Tg and the 3'-neighbor G*C base pair is disrupted. The solvent accessible surface and T(2) relaxation of Tg increases. Molecular dynamics calculations predict that the axial conformation of the Tg CH(3) 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.

Scheme 1.

(A) Interconversion of the cis-(5R,6S) and trans-(5R,6R) Tg lesions. When the 5R-Tg isomer is paired opposite dA in this 5′-GTgG-3′ sequence a 7:3 cis-(5R,6S): trans-(5R,6R) mixture is present at equilibrium, in slow exchange on the NMR timescale (12). (B) Oligodeoxynucleotide duplex used for NMR studies, indicating the numbering of the nucleotides. X⁶ is the cis-(5R,6S) Tg lesion.

Figure 1.

Binding of UvrA to the duplex containing the site-specific 5R-Tg lesion paired with dA. In (A) and (B), UvrA at the indicated concentrations was incubated with 2-nM substrates at 37°C for 20 min in UvrABC binding buffer and then analyzed on a 3.5% polyacrylamide native gel by gel mobility shift assays. (A) TG-51 bp; (B) AAF-50 bp. (C) The binding curves generated based on the titration data in (A) and (B).

Figure 2.

Incisions of the site-specific 5R-Tg paired with dA substrate by UvrABC nuclease. In (A) and (B), 2-nM DNA substrates 5′-terminally labeled with ³²P were incubated with UvrABC proteins in the UvrABC buffer in the presence of 1-mM ATP at 37°C for the indicated time periods. The incision products were analyzed on a 12% urea PAGE under denaturing conditions. (A) AAF-50 bp; (B) Tg-51 bp. (C) Kinetics of UvrABC incisions based upon the data in (A) and (B).

Figure 3.

Binding of human XPC/HR23B and XPA proteins to the site-specific 5R-Tg paired with dA substrate. In (A) and (B), XPA (A) or XPC/HR23B (B) proteins at the indicated concentrations were incubated with 4-nM substrates at 30°C for 30 min in XPA binding buffer and then analyzed on 3.5% polyacrylamide native gels by gel mobility shift assays (XPA, 4°C; XPC/HR23B, room temperature). (C) and (D) show the binding curves generated from the data in (A) and (B).

Figure 4.

(A) NOESY data collected at a NOE mixing time of 250 ms, showing NOEs between the cis-(5R,6S) Tg lesion and the DNA that were used for structural refinement. The spectra were collected at 800 MHz at a temperature of 30°C.

Figure 5.

Base pair stacking interactions of the cis-(5R,6S) Tg lesion. Comparison stacking interactions in which Tg CH₃ is in the axial (PDB ID 2KH5) or equatorial (PDB ID 2KH6) conformations. The top panel shows the G⁵• C²⁰ base pair (black) stacked above the X⁶• A¹⁹ base pair (Tg is colored red and A¹⁹ is colored gray). The center panel shows the orientation of the X⁶ lesion (red) with respect the complementary nucleotide A¹⁹ (black). The bottom panel shows the X⁶• A¹⁹ base pair (Tg is colored red and A¹⁹ is colored black) stacked above the G⁷• C¹⁸ base pair (grey).

Figure 6.

The cis-(5R,6S)Tg lesion at the X⁶• A¹⁹ base pair as viewed from the major groove showing potential hydrogen bonding interactions as predicted from analyses of rMD trajectories. (A) The Tg OH6 formed a hydrogen bond with G⁵ N7 when Tg CH₃ was in the axial conformation (PDB ID 2KH5). (B) When Tg CH₃ was in the equatorial conformation Tg OH6 did not hydrogen bond with G⁵ N7, however, improved hydrogen bonding was observed with Tg OH5 (PDB ID 2KH6).