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. 2009 Mar 13;386(5):1193-203.
doi: 10.1016/j.jmb.2008.12.082. Epub 2009 Jan 8.

The sequence dependence of human nucleotide excision repair efficiencies of benzo[a]pyrene-derived DNA lesions: insights into the structural factors that favor dual incisions

Konstantin Kropachev  1 , Marina Kolbanovskii, Yuqin Cai, Fabian Rodríguez, Alexander Kolbanovskii, Yang Liu, Lu Zhang, Shantu Amin, Dinshaw Patel, Suse Broyde, Nicholas E Geacintov
Affiliations

The sequence dependence of human nucleotide excision repair efficiencies of benzo[a]pyrene-derived DNA lesions: insights into the structural factors that favor dual incisions

Konstantin Kropachev et al. J Mol Biol. .

Abstract

Nucleotide excision repair (NER) is a vital cellular defense system against carcinogen-DNA adducts, which, if not repaired, can initiate cancer development. The structural features of bulky DNA lesions that account for differences in NER efficiencies in mammalian cells are not well understood. In vivo, the predominant DNA adduct derived from metabolically activated benzo[a]pyrene (BP), a prominent environmental carcinogen, is the 10S (+)-trans-anti-[BP]-N(2)-dG adduct (G*), which resides in the B-DNA minor groove 5'-oriented along the modified strand. We have compared the structural distortions in double-stranded DNA, imposed by this adduct, in the different sequence contexts 5'-...CGG*C..., 5'-...CG*GC..., 5'-...CIG*C... (I is 2'-deoxyinosine), and 5'-...CG*C.... On the basis of electrophoretic mobilities, all duplexes manifest moderate bends, except the 5'-...CGG*C...duplex, which exhibits an anomalous, slow mobility attributed to a pronounced flexible kink at the site of the lesion. This kink, resulting from steric hindrance between the 5'-flanking guanine amino group and the BP aromatic rings, both positioned in the minor groove, is abolished in the 5'-...CIG*C...duplex (the 2'-deoxyinosine group, I, lacks this amino group). In contrast, the sequence-isomeric 5'-...CG*GC...duplex exhibits only a moderate bend, but displays a remarkably increased opening rate at the 5'-flanking base pair of G*, indicating a significant destabilization of Watson-Crick hydrogen bonding. The NER dual incision product yields were compared for these different sequences embedded in otherwise identical 135-mer duplexes in cell-free human HeLa extracts. The yields of excision products varied by a factor of as much as approximately 4 in the order 5'-...CG*GC...>5'...CGG*C...>or=5'...CIG*C...>or=5'-...CG*C.... Overall, destabilized Watson-Crick hydrogen bonding, manifested in the 5'-...CG*GC...duplex, elicits the most significant NER response, while the flexible kink displayed in the sequence-isomeric 5'-...CGG*C...duplex represents a less significant signal in this series of substrates. These results demonstrate that the identical lesion can be repaired with markedly variable efficiency in different local sequence contexts that differentially alter the structural features of the DNA duplex around the lesion site.

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Figures

Figure 1
Figure 1
Structure and stereochemical properties of the 10S (+)-trans-anti-[BP]-N2-dG adduct (top). The base sequence contexts of the different duplexes studied and their abbreviations are defined here where G* = 10S (+)-trans-anti-[BP]-N2-dG. The NMR properties, , and the gel electrophoresis results (Figure 2) were obtained with the duplexes shown. The NER studies were conducted with the same duplexes embedded in 135-mer duplexes that otherwise had the same base sequence (Supporting Information).
Figure 2
Figure 2
Relative electrophoretic mobilities of unmodified and the 10S (+)-trans-anti-[BP]-N2-dG adduct-modified oligonucleotide duplexes defined in Figure 1 with their natural complementary strands in native 8% polyacrylamide gels at room temperature. The faint bands at the top of the gel represent the wells. Table: % decrease in electrophoretic mobilitya: (Mobility of the modified duplex – mobility of the unmodified duplex) × 100 / mobility of the unmodified duplex. bData from Xu et al.
Figure 3
Figure 3
(a) Comparison of nucleotide excision repair results in HeLa cell extracts for 135-mer duplexes containing the UV photodimer CPD or 6-4 duplexes, the 10S (+)-trans-anti-[BP]-N2-dG (CG*C), or the stereoisomeric (+)-cis adducts in the same CG*C sequence context (cis-CG*C). The (+)-cis-duplex was used as a reference standard in each set of experiments to adjust for variabilities in the NER activities of cell extracts prepared at different times (see text for details). The overall radioactivity levels in each lane were comparable, to allow for a visual inspection of the differences in dual incision efficiencies. Lanes 1, 2, 3, and 4 represent incubation times of 10, 20, 30, and 40 minutes respectively. The lane marked <TT> represents parallel incubations with unmodified <TT> duplexes, while lane M contains unmodified oligonucleotide markers 32, 30, 28, 26, and 24 nucleotides in length. In this experiment, the maximum extent of NER incisions (observed in the case of the 10R (+)-cis-anti-[BP]-N2-dG adduct in the cis-CG*C duplex at 40 min), was 4.6%. (b) Time dependence of formation of dual incision products. The average values and standard deviations are based on five independent experiments in the case of the two UV photodimers, and eight experiments in the case of the 10S (+)-trans-anti-[BP]-N2-dG adduct in the CG*C sequence. The straight lines are the least square fits to the data points, and the relative values of the slopes and the standard errors are summarized in Table S1, Supporting Information. In all these independent experiments, the data points for the the CPD <TT>, 6-4 <TT>, and CG*C (10S (+)-trans-anti-[BP]-N2-dG adduct) were normalized to the 40 min value of the 10R (+)-cis-anti-[BP]-N2-dG adduct-containing CG*C duplex (open circles) obtained in the same experiment. The inset in Figure 3b shows the densitometric tracings of the 40 min lanes shown in the gel (panel (a)).
Figure 4
Figure 4
Comparison of dual incisions elicited by the 10S (+)-trans-anti-[BP]-N2-dG adduct in CG*C, G6G7* (abbreviated as G7*), and G6*G7 (abbreviated as G7*) 135-mer duplexes in HeLa cell extracts after an incubation time of 40 min. Control samples: G6G7, unmodified duplex without or after treatment with cell extracts: CG*C, G6G7*, and G6*G7: untreated controls. The overall radioactivity levels in each lane were comparable, to allow for a visual inspection of the differences in dual incision efficiencies. Densitometry tracings of the lanes in panel comparing dual incision efficiencies of the same 10S (+)-trans-anti-[BP]-N2-dG adduct embedded in the different sequence contexts are shown in Figure S4, Supporting Information. The relative NER efficiency of the (+)-cis-anti-[BP]-N2-dG adduct used as a standard (See text) is also shown.
Figure 5
Figure 5
(a) Denaturing gel showing the appearance of dual excisions elicited by the 10S (+)-trans-anti-[BP]-N2-dG adducts in the I6G7*, G6*G7, G6G7* and CG*C duplexes as a function of incubation time in HeLa cell extracts. Lanes 1, 2, 3, and 4 represent incubation times of 10, 20, 30, and 40 min, respectively. Representative densitometry tracings, adjusted for the total radioactivity in each lane to compensate for loading factors and differences in the radioactivities of the samples (the concentration of the I6G7* was lower than in the case of the other samples). (b) Time course of dual incision product formation elicited by the 10S (+)-trans-anti-[BP]-N2-dG adduct in the G6*G7, G6G7*, I6G7* and CG*C duplexes. The experimental points are averages of seven independent experiments, and the error bars represent the standard deviations. The error bars for the I6G7* data points are omitted for clarity, but they do overlap with the error bars of the G6G7* and CG*C data points. The straight lines are the least square fits to the data points, and the relative values of the slopes and the standard error are summarized in Table S1, Supporting Information. In all these experiments, the data points were normalized to unity based on the 40 min value of the 10R (+)-cis-anti-[BP]-N2-dG in the CG*C duplex obtained in each individual experiment with a given cell extract (not shown). The lanes for the I6G7* duplex are uniformly lighter (panel a, left) than in the case of the other sequences; this was due to the lower quantities of I6G7* sample available. However, this difference is not reflected in panel b, since these data represent ratios of [dual excision products]/[total DNA substrates]) (see text).
Figure 6
Figure 6
Summary and comparisons of structural effects on relative NER efficiencies. Differential steric hindrance by nearby guanine amino groups governs sequence-dependent distortions and duplex destabilization, that influence the DNA sequence-governed NER dual incision efficiencies. In the series of minor groove lesion-containing duplexes studied here, the strongest signal that elicits the NER dual incision response is a disrupted Watson-Crick base pair in the G6*G7 duplex. The flexible kink in G6G7* appears to be less important since the bends are more rigid in I6G7* and the other two duplexes studied (see text). Minor groove enlargement, together with modest perturbation of Watson-Crick base pairing at the lesion site is common to all DNA sequences examined here.
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