Base sequence context effects on nucleotide excision repair

Abstract

Nucleotide excision repair (NER) plays a critical role in maintaining the integrity of the genome when damaged by bulky DNA lesions, since inefficient repair can cause mutations and human diseases notably cancer. The structural properties of DNA lesions that determine their relative susceptibilities to NER are therefore of great interest. As a model system, we have investigated the major mutagenic lesion derived from the environmental carcinogen benzo[a]pyrene (B[a]P), 10S (+)-trans-anti-B[a]P-N(2)-dG in six different sequence contexts that differ in how the lesion is positioned in relation to nearby guanine amino groups. We have obtained molecular structural data by NMR and MD simulations, bending properties from gel electrophoresis studies, and NER data obtained from human HeLa cell extracts for our six investigated sequence contexts. This model system suggests that disturbed Watson-Crick base pairing is a better recognition signal than a flexible bend, and that these can act in concert to provide an enhanced signal. Steric hinderance between the minor groove-aligned lesion and nearby guanine amino groups determines the exact nature of the disturbances. Both nearest neighbor and more distant neighbor sequence contexts have an impact. Regardless of the exact distortions, we hypothesize that they provide a local thermodynamic destabilization signal for repair.

Figure 1

(a) Chemical structure of the 10S (+)-trans-anti-B[a]P-N ²-dG adduct. (b) Base sequence contexts investigated. For 5′-…I[G*]C…, formally, inosine is the nucleoside, while hypoxanthine is the correct name for the corresponding base; for simplicity we utilize the term inosine.

Figure 2

Effects of nearby guanine amino groups on the positioning of the 10S (+)-trans-anti-B[a]P-N ²-dG adduct in the minor groove of the lesion-containing duplexes. The presence or absence and exact location of the guanine amino groups is governed by the base sequence contexts and determines the structural distortion/destabilization of the damaged duplexes. The damaged strand is light grey, and the partner is dark grey.

Figure 3

(a) In the 5′-…C[G*]G… sequence context, steric hinderance between the B[a]P moiety and nearby guanine amino groups causes the episodic denaturation of the C5 : G20 Watson-Crick hydrogen bond. (b) In the 5′-…G[G*]C… sequence context, steric hindrance between the B[a]P moiety and nearby guanine amino groups causes untwisting, manifested as a bend. (c) and (d) Definition of DNA duplex helicoidal parameters Roll and Twist, respectively. These cartoons are adapted with permission from Lu et al., Nucleic Acids Res. 31 (17): 5108–5121, Figure 1, Copyright 2003, Oxford University Press.

Figure 4

Hierarchy of NER recognition signals for the 10S (+)-trans-anti-B[a]P-N ²-dG adduct in various sequence contexts.

Figure 5

Cartoon representation of the lesion-containing duplexes (a) 5′-…C[G*]C-I… and (b) 5′-…C[G*]C-II…. Note that 5′-…C[G*]C-I… and 5′-…C[G*]C-II… differ beginning with the next nearest neighbor to [G*] and beyond. The locations of the key guanine amino group “1” and highly flexible dinucleotide C-A step “2”, present only in the 5′-…C[G*]C-II… sequence, are marked in red balloons.