UvrABC Incision Research Articles

Deciphering the essentiality and function of SxSx motif in Mycobacterium tuberculosis UvrB

The UvrB subunit is a central component of the UvrABC incision complex and plays a pivotal role in damage recognition, strand excision and repair synthesis. A conserved structural motif (the SxSx motif) present in UvrB is analogous to a similar motif (TxGx) in the helicases of superfamily 2, whose function is not fully understood. To elucidate the significance of the SxSx (Ser143-Val144-Ser145-Cys146) motif in Mycobacterium tuberculosis UvrB (MtUvrB), different variants of MtUvrB subunit were constructed and characterized. The SxSx motif indeed was found to be essential for MtUvrB function: while Ser143 and Cys146 residues within this motif were crucial for MtUvrB function, Ser145 plays an important but less essential role. The SxSx motif-deleted mutant was drastically attenuated and three single (S143A, S145A and C146A) mutants and a double (S143A/S145A) mutant exhibited various degrees of severity in their DNA-binding, DNA helicase and ATPase activities. Taken together, these results highlight a hitherto unrecognized role for SxSx motif in the catalytic activities of UvrB.

UvrA and UvrC subunits of the Mycobacteriumtuberculosis UvrABC excinuclease interact independently of UvrB and DNA.

The UvrABC excinuclease plays a vital role in bacterial nucleotide excision repair. While UvrA and UvrB subunits associate to form a UvrA2 B2 complex, interaction between UvrA and UvrC has not been demonstrated or quantified in any bacterial species. Here, using Mycobacteriumtuberculosis UvrA (MtUvrA), UvrB (MtUvrB) and UvrC (MtUvrC) subunits, we show that MtUvrA binds to MtUvrB and equally well to MtUvrC with submicromolar affinity. Furthermore, MtUvrA forms a complex with MtUvrC both invivo and invitro, independently of DNA and UvrB. Collectively, these findings reveal new insights into the pairwise relationships between the subunits of the UvrABC incision complex.

Correlation of Thermal Stability and Structural Distortion of DNA Interstrand Cross-Links Produced from Oxidized Abasic Sites with Their Selective Formation and Repair.

C4'-oxidized (C4-AP) and C5'-oxidized abasic sites (DOB) that are produced following abstraction of a hydrogen atom from the DNA backbone reversibly form cross-links selectively with dA opposite a 3'-adjacent nucleotide, despite the comparable proximity of an opposing dA. A previous report on UvrABC incision of DNA substrates containing stabilized analogues of the ICLs derived from C4-AP and DOB also indicated that the latter is repaired more readily by nucleotide excision repair [Ghosh, S., and Greenberg, M. M. (2014) Biochemistry 53, 5958-5965]. The source for selective cross-link formation was probed by comparing the reactivity of ICL analogues of C4-AP and DOB that mimic the preferred and disfavored cross-links with that of reagents that indirectly detect distortion by reacting with the nucleobases. The disfavored C4-AP and DOB analogues were each more reactive than the corresponding preferred cross-link substrates, suggesting that the latter are more stable, which is consistent with selective ICL formation. In addition, the preferred DOB analogue is more reactive than the respective C4-AP ICL, which is consistent with its more efficient incision by UvrABC. The conclusions drawn from the chemical probing experiments are corroborated by UV melting studies. The preferred ICLs exhibit melting temperatures higher than those of the corresponding disfavored isomers. These studies suggest that oxidized abasic sites form reversible interstrand cross-links with dA opposite the 3'-adjacent thymidine because these products are more stable and the thermodynamic preference is reflected in the transition states for their formation.

Nucleotide Excision Repair of Chemically Stabilized Analogues of DNA Interstrand Cross-Links Produced from Oxidized Abasic Sites

Nucleotide excision repair is a primary pathway in cells for coping with DNA interstrand cross-links (ICLs). Recently, C4′-oxidized (C4-AP) and C5′-oxidized abasic sites (DOB) that are produced following hydrogen atom abstraction from the DNA backbone were found to produce ICLs. Because some of the ICLs derived from C4-AP and DOB are too unstable to characterize in biochemical processes, chemically stable analogues were synthesized [Ghosh, S., and Greenberg, M. M. (2014) J. Org. Chem.79, 5948–5957]. UvrABC incision of DNA substrates containing stabilized analogues of the ICLs derived from C4-AP and DOB was examined. The incision pattern for the ICL related to the C4′-oxidized abasic site was typical for UvrABC substrates. UvrABC cleaved both strands of the substrate containing the C4-AP ICL analogue, but it was a poor substrate. UvrABC incised <30% of the C4-AP ICL analogue over an 8 h period, raising the possibility that this cross-link will be inefficiently repaired in cells. Furthermore, double-strand breaks were not detected upon incision of an internally labeled hairpin substrate containing the C4-AP ICL analogue. UvrABC incised the stabilized analogue of the DOB ICL more efficiently (∼20% in 1 h). Furthermore, the incision pattern was unique, and the cross-linked substrate was converted into a single product, a double-strand break. The template strand was exclusively incised on the template strand on the 3′-side of the cross-linked dA. Although the outcomes of the interaction between UvrABC and these two cross-linked substrates are different from one another, they provide additional examples of how seemingly simple lesions (C4-AP and DOB) can potentially exert significant deleterious effects on biochemical processes.

Repair of the Major Lesion Resulting from C5′-Oxidation of DNA

Oxidation of the C5'-position of DNA results in direct strand scission. The 3'-fragments produced contain DNA lesions at their 5'-termini. The major DNA lesion contains an aldehyde at its C5'-position, but its nucleobase is unmodified. Excision of the lesion formed from oxidation of thymidine (T-al) is achieved by strand displacement synthesis by DNA polymerase β (Pol β) in the presence or absence of flap endonuclease 1 (FEN1). Pol β displaces T-al and thymidine with comparable efficiency, but less so than a chemically stabilized abasic site analogue (F). FEN1 cleaves the flaps produced during strand displacement synthesis that are two nucleotides or longer. A ternary complex containing T-al is also a substrate for the bacterial UvrABC nucleotide excision repair system. The sites of strand scission are identical in ternary complexes containing T-al, thymidine, or F. UvrABC incision efficiency of these ternary complexes is comparable as well but significantly slower than a duplex substrate containing a bulky substituted thymidine. However, cleavage occurs only on the 5'-fragment and does not remove the lesion. These data suggest that unlike many lesions the redundant nature of base excision and nucleotide excision repair systems does not provide a means for removing the major damage product produced by agents that oxidize the C5'-position. This may contribute to the high cytotoxicity of drugs that oxidize the C5'-position in DNA.

Repair of mitomycin C mono- and interstrand cross-linked DNA adducts by UvrABC: a new model

Mitomycin C induces both MC-mono-dG and cross-linked dG-adducts in vivo. Interstrand cross-linked (ICL) dG-MC-dG-DNA adducts can prevent strand separation. In Escherichia coli cells, UvrABC repairs ICL lesions that cause DNA bending. The mechanisms and consequences of NER of ICL dG-MC-dG lesions that do not induce DNA bending remain unclear. Using DNA fragments containing a MC-mono-dG or an ICL dG-MC-dG adduct, we found (i) UvrABC incises only at the strand containing MC-mono-dG adducts; (ii) UvrABC makes three types of incisions on an ICL dG-MC-dG adduct: type 1, a single 5′ incision on 1 strand and a 3′ incision on the other; type 2, dual incisions on 1 strand and a single incision on the other; and type 3, dual incisions on both strands; and (iii) the cutting kinetics of type 3 is significantly faster than type 1 and type 2, and all of 3 types of cutting result in producing DSB. We found that UvrA, UvrA + UvrB and UvrA + UvrB + UvrC bind to MC-modified DNA specifically, and we did not detect any UvrB- and UvrB + UvrC–DNA complexes. Our findings challenge the current UvrABC incision model. We propose that DSBs resulted from NER of ICL dG-MC-dG adducts contribute to MC antitumor activity and mutations.

Abstract 1967: UvrABC excision of interstrand crosslink mitomycin C-DNA lesion induces double-stranded DNA breaks

Abstract Mitomycin C (MC) forms at least nine different covalent DNA adducts including an N2-2’-deoxyguanosine monoadduct (MC-mono-dG) and inter- and intrastrand bis-adducts with 2’-deoxyguanosine (dG) in vivo. Inter-strand cross-linked (ICL) dG-MC-dG-DNA adducts can prevent strand separation, and therefore, the MC antitumor activity is most likely derived from formation of ICL-MC adducts. In Escherichia coli cells, UvrABC, the nucleotide excision repair (NER) enzyme complex can repair ICL psoralen lesions that cause DNA bending. The mechanism and the consequence of NER of ICL dG-MC-dG lesions that do not induce DNA bending remain unclear. In order to compare the NER of MC-mono-dG with an ICL dG-MC-dG adduct, we constructed DNA fragments containing the adducts in the same DNA sequence context. We determined that UvrABC incises only at the strand containing MC-mono-dG adduct. In contrast, UvrABC makes three types of incisions on a site specific ICL dG-MC-dG adduct: type 1 (40 %), a single 5’ incision on one strand and 3’ incision on the other; type 2 (21 %), dual incisions on one strand and a single incision on the other strand; and type 3 (39%), dual incisions on both strand of both 5’ and 3’ sides of the ICL dG-MC-dG adduct. These three types of incisions occur with similar kinetics. An important consequence of these incisions is formation of DNA double-stranded breaks (DSB). Contrary to conventional model, we also found that UvrA, UvrA+UvrB and UvrA+UvrB+UvrC bind to MC-modified DNA specifically and form complexes that include UvrA. No UvrB- and UvrB+UvrCDNA complexes were observed. Our findings challenge the current UvrABC incision model of ICLs. We propose that NER of ICL dG-MC-dG adduct produces DSB, a lethal event that contributes to MC antitumor activity, and causes a non-homologous end-to-end joining that leads to DNA translocation and deletion mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1967.

Abstract 2986: Melanocytes are deficient in repair of oxidative DNA damage and UVC induced photoproducts

Abstract Melanomas occur mainly in sunlight-exposed skin, xeroderma pigmentosum complementation (XP) patients, defective in nucleotide excision repair function, have 1000-fold higher incidence of melanoma. These findings indicate that sunlight induced “bulky” photoproducts are responsible for melanomagenesis. On the other hand, sunlight also induces a high level of reactive oxygen species (ROS) in melanoctes (MC), oxidative DNA damage (ODD) therefore may contribute to melanomagenesis and that the XP protein may participate in ODD repair. To address these questions we determined: 1) the effect of melanin on UVA (320-400 nm) irradiation induced ODD and UV photoproducts using formamidopyrimidine glycosylase (Fpg) and UvrABC incision assays, 2) the repair capacity for ODD and UV-induced photoproducts in MC and XPA cells using host cell reactivation and in vitro DNA repair synthesis assays. We found: 1) UVA irradiation-inducing a significantly higher amount of Fpg sensitive ODD in MC than in normal human skin fibroblasts (NHSF). In contrast, UVA irradiation induces an insignificant amount of UvrABC sensitive sites in either of these two types of cells. 2) Compared to NHSF, MC have a reduced repair capacity for ODD and photoproducts. 3) H2O2 modified DNA induces a higher mutation frequency in MC than in NHSF. 4) XPA cells are deficient in ODD repair and ODD induces a higher mutation frequency in XPA cells than in NHSF. Based on these results, we propose: 1) UVA is a major etiological agent for melanomagenesis and the mechanism is through melanin sensitizes UVA in induction of ODD and reduced repair capacity in MC; 2) the reduced repair capacity for ODD contributes to the high melanoma incidence in XPA patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2986.

Benzene-derived N2-(4-hydroxyphenyl)-deoxyguanosine adduct: UvrABC incision and its conformation in DNA

Benzene, a ubiquitous human carcinogen, forms DNA adducts through its metabolites such as p-benzoquinone (p-BQ) and hydroquinone (HQ). N2-(4-Hydroxyphenyl)-2′-deoxyguanosine (N2-4-HOPh-dG) is the principal adduct identified in vivo by 32P-postlabeling in cells or animals treated with p-BQ or HQ. To study its effect on repair specificity and replication fidelity, we recently synthesized defined oligonucleotides containing a site-specific adduct using phosphoramidite chemistry. We here report the repair of this adduct by Escherichia coli UvrABC complex, which performs the initial damage recognition and incision steps in the nucleotide excision repair (NER) pathway. We first showed that the p-BQ-treated plasmid was efficiently cleaved by the complex, indicating the formation of DNA lesions that are substrates for NER. Using a 40-mer substrate, we found that UvrABC incises the DNA strand containing N2-4-HOPh-dG in a dose- and time-dependent manner. The specificity of such repair was also compared with that of DNA glycosylases and damage-specific endonucleases of E. coli, both of which were found to have no detectable activity toward N2-4-HOPh-dG. To understand why this adduct is specifically recognized and processed by UvrABC, molecular modeling studies were performed. Analysis of molecular dynamics trajectories showed that stable G:C-like hydrogen bonding patterns of all three Watson–Crick hydrogen bonds are present within the N2-4-HOPh-G:C base pair, with the hydroxyphenyl ring at an almost planar position. In addition, N2-4-HOPh-dG has a tendency to form more stable stacking interactions than a normal G in B-type DNA. These conformational properties may be critical in differential recognition of this adduct by specific repair enzymes.

Nucleotide Excision Repair of a DNA Interstrand Cross-Link Produces Single- and Double-Strand Breaks

The DNA radical resulting from formal abstraction of a hydrogen atom from the thymidine methyl group, 5-(2'-deoxyuridinyl)methyl radical, forms interstrand cross-links with the opposing 2'-deoxyadenosine. This is the first chemically characterized, radical-mediated cross-link between two opposing nucleotides. In addition, cross-linking between opposing bases in the duplex is less common than between those separated by one or two nucleotides. The first step in cross-link repair was investigated using the UvrABC bacterial nucleotide excision repair system. UvrABC incised both strands of the cross-linked DNA, although the strand containing the cross-linked purine was preferred by the enzyme in two different duplexes. The incision sites in one strand were spaced 11-14 nucleotides apart, as is typical for UvrABC incision. The majority of incisions occur at the third phosphate from the 3'-side of the cross-link and eighth or ninth phosphate on the 5'-side. In addition, cleavage was found to occur on both strands, producing double-strand breaks in approximately 25-29% of the incision events. This is the first example of double-strand cleavage during nucleotide excision repair of cross-linked DNA that does not already contain a strand break in the vicinity of the cross-link.

Acrolein is a major cigarette-related lung cancer agent: Preferential binding at p53 mutational hotspots and inhibition of DNA repair.

The tumor suppressor gene p53 is frequently mutated in cigarette smoke (CS)-related lung cancer. The p53 binding pattern of carcinogenic polycyclic aromatic hydrocarbons (PAHs) found in CS coincides with the p53 mutational pattern found in lung cancer, and PAHs have thus been considered to be major culprits for lung cancer. However, compared with other carcinogenic compounds, such as aldehydes, the amount of PAHs in CS is minute. Acrolein (Acr) is abundant in CS, and it can directly adduct DNA. Acr-DNA adducts, similar to PAH-DNA adducts, induce predominantly G-to-T transversions in human cells. These findings raise the question of whether Acr-DNA adducts are responsible for p53 mutations in CS-related lung cancer. To determine the role of Acr-DNA adducts in p53 mutagenesis in CS-related lung cancer we mapped the distribution of Acr-DNA adducts at the sequence level in the p53 gene of lung cells using the UvrABC incision method in combination with ligation-mediated PCR. We found that the Acr-DNA binding pattern is similar to the p53 mutational pattern in human lung cancer. Acr preferentially binds at CpG sites, and this enhancement of binding is due to cytosine methylation at these sequences. Furthermore, we found that Acr can greatly reduce the DNA repair capacity for damage induced by benzo[a]pyrene diol epoxide. Together these results suggest that Acr is a major etiological agent for CS-related lung cancer and that it contributes to lung carcinogenesis through two detrimental effects: DNA damage and inhibition of DNA repair.

Base Flipping in Nucleotide Excision Repair

UvrB, the ultimate damage-binding protein in bacterial nucleotide excision repair is capable of binding a vast array of structurally unrelated lesions. A beta-hairpin structure in the protein plays an important role in damage-specific binding. In this paper we have monitored DNA conformational alterations in the UvrB-DNA complex, using the fluorescent adenine analogue 2-aminopurine. We show that binding of UvrB to a DNA fragment with cholesterol damage moves the base adjacent to the lesion at the 3' side into an extrahelical position. This extrahelical base is not accessible for acrylamide quenching, suggesting that it inserts into a pocket of the UvrB protein. Also the base opposite this flipped base is extruded from the DNA helix. The degree of solvent exposure of both residues varies with the type of cofactor (ADP/ATP) bound by UvrB. Fluorescence of the base adjacent to the damage is higher when UvrB is in the ADP-bound configuration, but concomitantly this UvrB-DNA complex is less stable. In the ATP-bound form the UvrB-DNA complex is very stable and in this configuration the base in the non-damaged strand is more exposed. Hairpin residue Tyr-95 is specifically involved in base flipping in the non-damaged strand. We present evidence that this conformational change in the non-damaged strand is important for 3' incision by UvrC.

Effects of DNA adduct structure and sequence context on strand opening of repair intermediates and incision by UvrABC nuclease.

DNA damage recognition of nucleotide excision repair (NER) in Escherichia coli is achieved by at least two steps. In the first step, a helical distortion is recognized, which leads to a strand opening at the lesion site. The second step involves the recognition of the type of chemical modification in the single-stranded region of DNA during the processing of the lesions by UvrABC. In the current work, by comparing the efficiencies of UvrABC incision of several types of different DNA adducts, we show that the size and position of the strand opening are dependent on the type of DNA adducts. Optimal incision efficiency for the C8-guanine adducts of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) was observed in a bubble of three mismatched nucleotides, whereas the same for C8-guanine adduct of 1-nitropyrene and N(2)-guanine adducts of benzo[a]pyrene diol epoxide (BPDE) was noted in a bubble of six mismatched nucleotides. This suggests that the size of the aromatic ring system of the adduct might influence the extent and number of bases associated with the opened strand region catalyzed by UvrABC. We also showed that the incision efficiency of the AF or AAF adduct was affected by the neighboring DNA sequence context, which, in turn, was the result of differential binding of UvrA to the substrates. The sequence context effect on both incision and binding disappeared when a bubble structure of three bases was introduced at the adduct site. We therefore propose that these effects relate to the initial step of damage recognition of DNA structural distortion. The structure-function relationships in the recognition of the DNA lesions, based on our results, have been discussed.

The effect of C(5) cytosine methylation at CpG sequences on mitomycin–DNA bonding profiles

Recent studies have documented that cytosine C(5) methylation of CpG sequences enhances mitomycin C (1) adduction. The reports differ on the extent and uniformity of 1 modification at the nucleotide level. We have determined the bonding profiles for mitomycin monoalkylation in two DNA restriction fragments where the CpG sequences were methylated. Three mitomycin substrates were used and two different enzymatic assays employed to monitor the extent of drug modification at the individual base sites. Drug–DNA modification was accomplished with 1 and 10-decarbamoylmitomycin C (2) under reductive (Na2S2O4) conditions and with N-methyl-7-methoxyaziridinomitosene (3) under nonreductive conditions. The UvrABC incision assay permitted us to quantitate the sites of drug adduction, and the λ-exonuclease stop assay provided a qualitative estimation of drug–DNA modification consistent with the UvrABC data. We learned that C(5) cytosine methylation (m5C) enhanced the extent of overall DNA modification. Using the UvrABC endonuclease assay, we found that modification by 1 increased 2.0 and 7.4 times for the two DNA restriction fragments. Analysis of the modification sites at the nucleotide sequence level revealed that guanine (G) was the only base modified and that the overall increased level of DNA adduction was due to enhanced modification of select m5CpG* (G*=mitomycin (mitosene) adduction sites) loci compared with CpG* sites: the largest differences reached two orders of magnitude. Significantly, not all CpG* sites underwent increased drug adduction upon C(5) cytosine methylation. The effect of C(5) cytosine methylation on the drug adduction profiles was less pronounced for G* sites located within dinucleotide sequences other than CpG*. We observed that DNA methylation often led to slightly diminished adduction levels at these sites. The different m5CpG* adduction patterns provided distinctive sequence-selective bonding profiles for 1–3. We have attributed the large differences in guanine reactivity to DNA structural factors created, in part, by C(5) cytosine methylation. The significance of these findings in cancer chemotherapy is briefly discussed.

Use of UvrABC nuclease to quantify benzo[a]pyrene diol epoxide-DNA adduct formation at methylated versus unmethylated CpG sites in the p53 gene.

We have used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction (LMPCR) techniques to map and quantify (+/-)anti-7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (BPDE) adduct formation in the p53 gene of human cells. We found that BPDE adduct formation, as revealed by UvrABC incision, preferentially occurred at methylated CpG sites that correspond to the mutational hotspots observed in human lung cancers. Our hypothesis is that it is this methylated CpG sequence-dependent preferential adduct formation, rather than selective growth advantage, that is the major determinant of the p53 mutation pattern in human cancers. Given the far reaching ramifications of such conclusions for cancer etiology, a legitimate question is raised regarding the reliability of using the UvrABC incision method for quantifying and determining the sequence-dependency of adduct formation. Is the higher frequency of UvrABC cutting at methylated versus unmethylated CpG sites due to the preference of the nuclease for cutting at those sites or due to the preferential formation of BPDE adducts at those sites? In order to distinguish between these two possibilities, we have analyzed the kinetics of UvrABC incision at BPDE adducts formed at either methylated CpG sites versus other sequences, or unmethylated CpG sites versus other sequences in exon 5 of the p53 gene. We have found that the UvrABC cutting kinetics are identical for both cases. On the basis of these results we conclude that under proper cutting conditions, UvrABC nuclease reacts with and incises with equal efficiency, BPDE adducts formed at methylated or unmethylated CpG sites as well as other sequences, and that the extent of UvrABC incision accurately reflects the extent of BPDE-DNA adduct formation. These conclusions were further supported by results obtained using a DNA synthesis blockage assay.

Slow repair of bulky DNA adducts along the nontranscribed strand of the human p53 gene may explain the strand bias of transversion mutations in cancers.

Using UvrABC incision in combination with ligation-mediated PCR (LMPCR) we have previously shown that benzo(a)pyrene diol epoxide (BPDE) adduct formation along the nontranscribed strand of the human p53 gene is highly selective; the preferential binding sites coincide with the major mutation hotspots found in human lung cancers. Both sequence-dependent adduct formation and repair may contribute to these mutation hotspots in tumor tissues. To test this possibility, we have extended our previous studies by mapping the BPDE adduct distribution in the transcribed strand of the p53 gene and quantifying the rates of repair for individual damaged bases in exons 5, 7, and 8 for both DNA strands of this gene in normal human fibroblasts. We found that: (i) on both strands, BPDE adducts preferentially form at CpG sequences, and (ii) repair of BPDE adducts in the transcribed DNA strand is consistently faster than repair of adducts in the nontranscribed strand, while repair at the major damage hotspots (guanines at codons 157, 248 and 273) in the nontranscribed strand is two to four times slower than repair at other damage sites. These results strongly suggest that both preferential adduct formation and slow repair lead to hotspots for mutations at codons 157, 248 and 273, and that the strand bias of bulky adduct repair is primarily responsible for the strand bias of G to T transversion mutations observed in the p53 gene in human cancers.

Detection of DNA damage by Escherichia coli UvrB-binding competition assay is limited by the stability of the UvrB-DNA complex.

To investigate the use of UvrB-binding to detect DNA damage, mobility shift gel electrophoresis was used to detect binding of UvrB protein to a 136 bp DNA fragment that was randomly adducted with aflatoxin B1 8,9-epoxide and end-labelled with 32P. After polyacrylamide gel electrophoresis, the shifted band that contained DNA bound by UvrB was quantified as a percentage of total radioactive substrate DNA. This method was applied to analyse plasmid DNA that was adducted with various DNA modifying agents in vitro. These adducts competed for UvrB-binding to the labelled substrate. By competing for UvrB-binding with 10 ng of plasmid DNA that was adducted with known levels of aflatoxin B1, 2-amino-3-methylimidazo[4,5-f]quinoline, or benzo[a]pyrene diol epoxide, UvrB competition could be quantified for DNA adducted with between one adduct in 10(2) and one adduct in 10(5) normal nucleotides. However, plasmid DNA exposed to N-methyl-N-nitrosourea or methylene blue + visible light, did not compete for UvrB-binding, even though the presence of UvrABC sensitive sites were confirmed on this DNA by a UvrABC incision assay. Mono-adducted 96-bp DNA substrates, which contained an internal 32P-label and either a single apurinic site, aflatoxin B1-guanine adduct, O6-methylguanine, 8-oxo-deoxyguanosine or non-adducted guanine, were also used as substrates for UvrA- and UvrB-binding to examine the stability of UvrB-DNA complexes with specific adducts. Under similar conditions used for the competition assay, significant UvrB-binding was seen only for the aflatoxin adducted substrate. These results suggest that stability of UvrB-binding varies greatly between bulky and non-bulky adducts. It was also found that rat liver DNA from untreated rats inhibited UvrB-binding to the substrate DNA in the competition assay, to a degree that was equivalent to competition with plasmid adducted at one adduct in 10(3) normal nucleotides.

Differential human nucleotide excision repair of paired and mispaired cisplatin-DNA adducts.

In order to understand the action of the chemotherapeutic drug cisplatin, it is necessary to determine why some types of cisplatin-DNA intrastrand crosslinks are repaired better than others. Using cell extracts and circular duplex DNA, we compared nucleotide excision repair of uniquely placed 1,2-GG, 1,2-AG, and 1,3-GTG cisplatin-crosslinks, and a 2-acetylaminofluorene lesion. The 1,3 crosslink and the acetylaminofluorene lesion were repaired by normal cell extracts approximately 15-20 fold better than the 1,2 crosslinks. No evidence was found for selective shielding of 1,2 cisplatin crosslinks from repair by cellular proteins. Fractionation of cell extracts to remove putative shielding proteins did not improve repair of the 1,2-GG crosslink, and cell extracts did not selectively inhibit access of UvrABC incision nuclease to 1,2-GG crosslinks. The poorer repair of 1,2 crosslinks in comparison to the 1,3 crosslink is more likely a consequence of different structural alterations of the DNA helix. In support of this, a 1,2-GG-cisplatin crosslink was much better repaired when it was opposite one or two non-complementary thymines. Extracts from cells defective in the hMutSalpha mismatch binding activity also showed preferential repair of the 1,3 crosslink over the 1,2 crosslink, and increased repair of the 1,2 adduct when opposite thymines, showing that hMutSalphais not involved in the differential NER of these substrates in vitro. Mismatched cisplatin adducts could arise by translesion DNA synthesis, and improved repair of such adducts could promote cisplatin-induced mutagenesis in some cases.

Sequence Preference of 7,12-Dimethylbenz[a]anthracene-syn-diol Epoxide−DNA Binding in the Mouse H-ras Gene Detected by UvrABC Nucleases

We have found that 7,12-dimethylbenz[a]anthracene-syn-diol epoxide (syn-DMBADE)-modified DNA fragments are sensitive to UvrABC incision. The incisions occur mainly seven bases 5' and four bases 3' of a syn-DMBADE-modified adenine or guanine residue. The kinetics of UvrABC incision at different sequences in a DNA fragment are the same, and the extent of UvrABC incision is proportional to the syn-DMBADE concentration. On the basis of these results, we have concluded that UvrABC incision on syn-DMBADE-DNA adducts is independent of DNA sequence and is quantitative. Using the UvrABC incision method, we have analyzed the syn-DMBADE-DNA binding spectrum in several defined DNA fragments, including the first two exons of the mouse H-ras gene. We have found that both guanine and adenine residues in codons 12, 13, and 61 of the H-ras gene are strong syn-DMBADE binding sites. These results suggest that the initial binding of DMBADE may greatly contribute to the frequency of H-ras mutations. Results from dinucleotide binding analysis indicate that the 5'-nearest neighbor displays a greater effect on syn-DMBADE-DNA binding than the 3'-nearest neighbor.

Using UvrABC nuclease to detect 7,12-dimethylbenz[a]anthracene anti-diol epoxide-DNA binding specificity in the mouse H-ras gene.

DNA fragments modified with chemically synthesized 7,12-dimethylbenz[a]anthracene anti-diol epoxide (anti-DMBADE) are sensitive to UvrABC nuclease incision. The incisions occur mainly 7 bases 5' and 4 bases 3' of an anti-DMBADE-modified adenine or guanine residue, and the kinetics of incision at different sequences in a DNA fragment are the same. These results indicate that UvrABC incision on anti-DMBADE-DNA adducts is independent of DNA sequences and is quantitative, the same as on syn-DMBADE-DNA adducts. This method was used to analyze the anti-DMBADE-DNA binding spectrum in the exon 2 region of the mouse H-ras gene, and it was found that anti-DMBADE binds to the two adenine residues at codon 61 of the H-ras gene with an average affinity. Previously, we have demonstrated that syn-DMBADE binds strongly to the adenines at codon 61 of H-ras; these results together suggest that the oncogenic mutation in H-ras may be induced by anti- and syn-DMBADE-DNA adducts.