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Comparative Study
. 2011 Jul 15;10(7):684-96.
doi: 10.1016/j.dnarep.2011.04.020. Epub 2011 Jul 8.

Probing for DNA damage with β-hairpins: similarities in incision efficiencies of bulky DNA adducts by prokaryotic and human nucleotide excision repair systems in vitro

Yang Liu  1 , Dara Reeves, Konstantin Kropachev, Yuqin Cai, Shuang Ding, Marina Kolbanovskiy, Alexander Kolbanovskiy, Judith L Bolton, Suse Broyde, Bennett Van Houten, Nicholas E Geacintov
Affiliations
Comparative Study

Probing for DNA damage with β-hairpins: similarities in incision efficiencies of bulky DNA adducts by prokaryotic and human nucleotide excision repair systems in vitro

Yang Liu et al. DNA Repair (Amst). .

Abstract

Nucleotide excision repair (NER) is an important prokaryotic and eukaryotic defense mechanism that removes a large variety of structurally distinct lesions in cellular DNA. While the proteins involved are completely different, the mode of action of these two repair systems is similar, involving a cut-and-patch mechanism in which an oligonucleotide sequence containing the lesion is excised. The prokaryotic and eukaryotic NER damage-recognition factors have common structural features of β-hairpin intrusion between the two DNA strands at the site of the lesion. In the present study, we explored the hypothesis that this common β-hairpin intrusion motif is mirrored in parallel NER incision efficiencies in the two systems. We have utilized human HeLa cell extracts and the prokaryotic UvrABC proteins to determine their relative NER incision efficiencies. We report here comparisons of relative NER efficiencies with a set of stereoisomeric DNA lesions derived from metabolites of benzo[a]pyrene and equine estrogens in different sequence contexts, utilizing 21 samples. We found a general qualitative trend toward similar relative NER incision efficiencies for ∼65% of these substrates; the other cases deviate mostly by ∼30% or less from a perfect correlation, although several more distant outliers are also evident. This resemblance is consistent with the hypothesis that lesion recognition through β-hairpin insertion, a common feature of the two systems, is facilitated by local thermodynamic destabilization induced by the lesions in both cases. In the case of the UvrABC system, varying the nature of the UvrC endonuclease, while maintaining the same UvrA/B proteins, can markedly affect the relative incision efficiencies. These observations suggest that, in addition to recognition involving the initial modified duplexes, downstream events involving UvrC can also play a role in distinguishing and processing different lesions in prokaryotic NER.

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Figures

Figure 1
Figure 1
(A) Crystal structure of Rad4-Rad23, the yeast homologue of human XPC-RAD23B. (PDB ID [74]: 2QSG) [23]; The two flipped-out thymines (red) in the unmodified strand are mismatched with the thymines of the T<>T photodimer, and are within interaction distances of amino acid residues in Rad4. The β-hairpin loop (marine) is inserted between the two strands, thus anchoring Rad4 (or XPC) to the site of the damage. (B) Model of UvrB [35] based on a UvrB-DNA co-crystal structure (PDB ID [74]: 2FDC) [25] with the DNA extended and a (+)-cis-BP-derived adduct (designated by *) modeled into a conserved pocket, concealed behind the β-hairpin. Color code: β-hairpin, marine; flipped-out bases, red. The figures were prepared using the PyMOL Molecular Graphics System [75].
Figure 2
Figure 2
(A) Sequence contexts and (B) chemical structures of the DNA adducts studied. Subscripted numbers designate the positions of the modified nucleotides (only one lesion per oligonucleotide). In sequences I, VI and VII the labeled adenine and cytosine bases denote 4-OHEN-dA or –dC adducts, respectively. The labeled guanine residues (G6) in sequence I and II (Del) denote either trans- or cis-BP-dG adducts derived from the reactions of (+)-anti-BPDE with the single-stranded sequence I. In sequences III, IV and V, G6 denotes the trans-BP-dG adduct. The abbreviations used for these adducts in the text are shown in square brackets. The full sequences of the oligonucleotide 135-mers containing the indicated duplexes used in HeLa cell NER experiments are defined elsewhere [15], and those of the shorter duplexes used in the UvrABC NER experiments are shown in Supporting Information.
Figure 3
Figure 3
NMR solution structures of the DNA adducts studied. The trans-BP-dG adduct in the full duplex (Duplex I) has the pyrenyl ring system positioned in the B-DNA minor groove directed 5'-along the modified strand with only minor perturbations of Watson-Crick base pairing [61]. In the cis-BP-dG full duplex I [57] and the deletion duplex [58], the conformation is base-displaced/intercalated, with the pyrenyl ring system inserted into the duplex. In the full duplex I, the modified G and its partner C are displaced into the minor groove and major groove, respectively, and the benzylic ring is directed toward the minor groove. In the (+)-cis-BP-dG duplex II (Del), the structure is very similar except that the partner C to the modified G is missing. However, the conformation of the trans-BP-dG duplex II (Del) differs from that of the full duplex in that it is base-displaced/intercalated, the modified G is displaced into the major groove, and the benzylic ring is directed toward the major groove [76]. In the (−)-SRR-4-OHEN-dC and the (+)-RSS-4-OHEN-dC adducts (Duplexes I and VI), the modified dC is in the anti-glycosidic bond conformation, the equilenin rings are in the minor groove with the distal rings protruding through the helix to the major groove; the rings are directed 5' along the modified strand in the case of (−)-SRR-4-OHEN-dC and 3'-oriented in the case of the (+)-RSS-4-OHEN-dC adduct [70].
Figure 4
Figure 4
(A) Autoradiogram of typical incisions catalyzed by UvrABcaBBcaCTma at 55 °C using the cis- and trans-BP-dG lesions embedded in the sequence contexts of duplexes I or II (Del) positioned in the middle of otherwise identical 135-mer DNA duplexes; these duplexes were 32P-endlabeled at the 6th phosphodiester bond on the 5'-side of the adduct. Lane M shows the migration distances of oligonucleotide markers 8, 10, 12, 14……28, 32 nucleotides in length (the dark band near the middle represents a 20-mer). The dual incision products migration distance is intermediate between those of the 12 and 14-nucleotide markers. (B) Kinetics of accumulation of incision products as a function of incubation time. Time dependence of formation of dual incision products. The average values and standard deviations are based on seven independent experiments of this type.
Figure 5
Figure 5
Comparisons of relative initial incision rates measured in human HeLa cell extracts and catalyzed by UvrABC proteins. In all cases, one of the substrates was assigned a value of 100, and the initial incision rates of the other substrates are expressed relative to the value of 100. The sources of the data are cited below in each case. (A) Effects of adduct stereochemistry and conformation: cis- and trans-BP-dG in the full duplexes I and deletion duplexes II (Del); NER data obtained using HeLa cell extracts and UvrABcaBBcaCTma at 55 °C. The HeLa data is from [36, 56], and the prokaryotic data is from Figure 4. (B) Effects of base sequence context, …TG6T… vs. …CG6C… with G6 denoting the trans-BP-dG adducts in duplexes III and IV (UvrABcaBBcaCBca at 37 °C, 43-mer 5'-endlabeled duplexes). The HeLa data is from [59] and the UvrABC data is from [9]. (C) Effects of base sequence context (G6 or G7) in …CG6G…, …GG7C…, or …CG6C… in sequence contexts V, V, and I, respectively, containing trans-BP-dG adducts. The HeLa data are from [15]. The UvrABC data for UvrABcaBBcaCBca at 55 °C are from Figure S1, Supporting Information (average and standard deviations from four independent experiments using 54-mer duplexes; the sequence is shown in Scheme 2).
Figure 6
Figure 6
Impact of different UvrC endonucleases on relative initial incision rates catalyzed by UvrABcaUvrBBcaUvrCBca, UvrABcaUvrBBcaUvrCTma, or UvrABcaUvrBBcaUvrCCho proteins of trans-BP-dG adducts in duplex V embedded in 5'-endlabeled 54-mer duplexes. The original data for UvrABcaBBcaCBca and UvrABcaUvrBBcaUvrCCho experiments are presented in Figures S2 and S3, respectively (Supporting Information), while the UvrABcaUvrBBcaUvrCTma data is from [77]. The HeLa cell extract results shown are from Figure 5C.
Figure 7
Figure 7
Incisions of stereoisomeric 4-OHEN-dC adducts at either position C5 or C6 in sequence I embedded in 5'-end-labeled 43-mer duplexes by E.coli UvrABC proteins at 37 °C (the full sequence is depicted in Scheme 3, Supporting Information). (A) Typical gel autoradiogram. (B) Incision kinetics. The average values are based on three independent experiments (data from Chen [41]). The minus and plus signs correspond, respectively, to the (−)-SRR- or the (+)-RSS-4-OHEN-dC adducts (Figure 2). (C).Comparison of relative initial rates of incisions of stereoisomeric 4-OHEN-dC6 and 4-OHEN-dC7 adducts in the duplex I sequence context embedded in either (1) the middle of 135-mer duplexes incubated in HeLa cell extracts and 32P-internally labeled at the 6th phosphodiester bond on the 5'-side of the adduct, and (2) in the middle of 5'-endlabeled 43-mer duplexes (See Supporting Information Scheme 2) catalyzed by E. coli UvrABC (these data were obtained from the initial linear portions of the plots in Figure 7B).
Figure 8
Figure 8
Comparisons of relative NER incision efficiencies of different stereoisomeric 4-OHEN-dC or –dA adducts in sequence contexts VI or VII embedded in the middle of internally labeled 135-mer DNA duplexes (See Supporting Information Scheme 1), in human HeLa cell extracts and prokaryotic UvrABcaBBcaCTma proteins at 55 °C. The value of 100 was assigned to the (+)-4-OHEN-dC6 adducts in sequence context VI (abbreviated as the (+)-dC6-VI adduct in the Figure), and all other incision efficiencies are expressed relative to this value. The HeLa and UvrABC data (typical gels are shown in Figure S4, Supporting Information), are excerpted from Reeves [42]. The minus and plus signs denote the stereochemical properties of the adducts (Figure 2), and the subscripts denote the positions of the modified nucleotides in sequences VI or VII.
Figure 9
Figure 9
Comparisons of all relative incision efficiencies in human HeLa cell extracts and UvrABC experiments shown in the individual bar graphs in Figures 5, 6, 7, and 8. The origins of each of the points are also shown. Incision efficiencies for each figure are expressed relative to a designated adduct assigned a value of 100. The straight line corresponds to a hypothetical ideal correlation; any point on this line would signify that the relative incision efficiencies are identical in both NER systems. A correlation coefficient of 0.595 was calculated for all the data points.
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