Evidence for Escherichia coli polymerase II mutagenic bypass of intrastrand DNA crosslinks

The mutagenic potentials of DNAs containing site- and stereospecific intrastrand DNA crosslinks were evaluated in Escherichia coli cells that contained a full complement of DNA polymerases or were deficient in either polymerases II, IV, or V. Crosslinks were made between adjacent N(6)-N(6) adenines and consisted of R,R- and S,S-butadiene crosslinks and unfunctionalized 2-, 3-, and 4-carbon tethers. Although replication of single-stranded DNAs containing the unfunctionalized 3- and 4-carbon tethers were non-mutagenic in all strains tested, replication past all the other intrastrand crosslinks was mutagenic in all E. coli strains, except the one deficient in polymerase II in which no mutations were ever detected. However, when mutagenesis was analyzed in cells induced for SOS, mutations were not detected, suggesting a possible change in the overall fidelity of polymerase II under SOS conditions. These data suggest that DNA polymerase II is responsible for the in vivo mutagenic bypass of these lesions in wild-type E. coli.     

Robust incision of Benoz[a]pyrene-7,8-dihyrodiol-9,10-epoxide-DNA adducts by a recombinant thermoresistant interspecies combination UvrABC endonuclease system

Prokaryotic DNA repair nucleases are useful reagents for detecting DNA lesions. UvrABC endonuclease, encoded by the UvrA, UvrB, and UvrC genes can incise DNA containing bulky nucleotide adducts and intrastrand cross-links. UvrA, UvrB, and UvrC were cloned from Bacillus caldotenax (Bca)and UvrC from Thermatoga maritima (Tma), and recombinant proteins were overexpressed in and purified from Escherichia coli. Incision activities of UvrABC composed of all Bca-derived subunits (UvrABC(Bca)) and an interspecies combination UvrABC composed of Bca-derived UvrA and UvrB and Tma-derived UvrC (UvrABC(Tma)) were compared on benoz[a]pyrene-7,8-dihyrodiol-9,10-epoxide (BPDE)-adducted substrates. Both UvrABC(Bca) and UvrABC(Tma) specifically incised both BPDE-adducted plasmid DNAs and site-specifically modified 50-bp oligonucleotides containing a single (+)-trans- or (+)-cis-BPDE adduct. Incision activity was maximal at 55-60 degrees C. However, UvrABC(Tma) was more robust than UvrABC(Bca) with 4-fold greater incision activity on BPDE-adducted oligonucleotides and 1.5-fold greater on [(3)H]BPDE-adducted plasmid DNAs. Remarkably, UvrABC(Bca) incised only at the eighth phosphodiester bond 5' to the BPDE-modified guanosine. In contrast, UvrABC(Tma) performed dual incision, cutting at both the fifth phosphodiester bond 3' and eighth phosphodiester bond 5' from BPDE-modified guanosine. BPDE adduct stereochemistry influenced incision activity, and cis adducts on oligonucleotide substrates were incised more efficiently than trans adducts by both UvrABC(Bca) and UvrABC(Tma). UvrAB-DNA complex formation was similar with (+)-trans- and (+)-cis-BPDE-adducted substrates, suggesting that UvrAB binds both adducts equally and that adduct configuration modifies UvrC recognition of the UvrAB-DNA complex. The dual incision capabilities and higher incision activity of UvrABC(Tma) make it a robust tool for DNA adduct studies.     

Translesion DNA synthesis by yeast DNA polymerase eta on templates containing N2-guanine adducts of 1,3-butadiene metabolites

Yeast DNA polymerase eta can replicate through cis-syn cyclobutane pyrimidine dimers and 8-oxoguanine lesions with the same efficiency and accuracy as replication of an undamaged template. Previously, it has been shown that Escherichia coli DNA polymerases I, II, and III are incapable of bypassing DNA substrates containing N(2)-guanine adducts of stereoisomeric 1,3-butadiene metabolites. Here we showed that yeast polymerase eta replicates DNA containing the monoadducts (S)-butadiene monoepoxide and (S,S)-butadiene diolepoxide N(2)-guanines albeit at an approximately 200-300-fold lower efficiency relative to the control guanine. Interestingly, nucleotide incorporation opposite the (R)-butadiene monoepoxide and the (R,R)-butadiene diolepoxide N(2)-guanines was approximately 10-fold less efficient than incorporation opposite their S stereoisomers. Polymerase eta preferentially incorporates the correct nucleotide opposite and downstream of all four adducts, except that it shows high misincorporation frequencies for elongation of C paired with (R)-butadiene monoepoxide N(2)-guanine. Additionally, polymerase eta does not bypass the (R,R)- and (S,S)-butadiene diolepoxide N(2)-guanine-N(2)-guanine intra- strand cross-links, and replication is completely blocked just prior to the lesion. Collectively, these data suggest that polymerase eta can tolerate the geometric distortions in DNA conferred by the N(2)-guanine butadiene monoadducts but not the intrastrand cross-links.     

In vitro repair of psoralen-DNA cross-links by RecA, UvrABC, and the 5'-exonuclease of DNA polymerase I

Psoralens produce DNA interstrand cross-links which are thought to be repaired via a sequential excision and recombination mechanism in Escherichia coli. The first round of incision by UvrABC has been characterized: it results in 11-base oligonucleotide cross-linked to an intact DNA strand (Van Houten, B., Gamper, B., Holbrook, S.R., Hearst, J.E., and Sancar, A. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8077-8081). In the present work, DNA substrates containing 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) cross-links in defined positions are constructed and used to analyze the other steps in repair. It is shown that RecA protein mediates strand transfer past an oligonucleotide cross-linked to a single-stranded DNA circle and that the resulting heteroduplex is a substrate for the UvrABC complex: it excises a double-stranded oligonucleotide which contains the HMT cross-link. It is also found that the first round of UvrABC incision does not lead directly to strand exchange but that an intervening step is needed. That step is carried out in vitro by the 5'-exonuclease activity of DNA polymerase I (pol I) which creates a single-stranded DNA region (a gap) at an incised cross-link such that RecA can initiate strand exchange. Studies using cross-linked oligonucleotides showed that the gap produced by pol I results from the inability of the polymerase to add nucleotides to a 3'-OH end two to three nucleotides away from the furan side of an HMT cross-link. Pol I can, however, extend a 3'-OH end next to the pyrone side of the cross-link. Since UvrABC incises predominantly the furan side of psoralen cross-links in duplex DNA, this discrepancy has important consequences for repair.     

Hierarchy of DNA damage recognition in Escherichia coli nucleotide excision repair

DNA damage recognition plays a central role in nucleotide excision repair (NER). Here we present evidence that in Escherichia coli NER, DNA damage is recognized through at least two separate but successive steps, with the first focused on distortions from the normal structure of the DNA double helix (initial recognition) and the second specifically recognizing the type of DNA base modifications (second recognition), after an initial local separation of the DNA strands. DNA substrates containing stereoisomeric (+)- or (-)-trans- or (+)- or (-)-cis-BPDE-N(2)-dG lesions in DNA duplexes of known conformations were incised by UvrABC nuclease with efficiencies varying by up to 3-fold. However, these stereoisomeric adducts, when positioned in an opened, single-stranded DNA region, were all incised with similar efficiencies and with enhanced rates (by factors of 1.4-6). These bubble substrates were also equally and efficiently incised by UvrBC nuclease without UvrA. Furthermore, removal of the Watson-Crick partner cytosine residue (leaving an abasic site) in the complementary strand opposite a (+)-cis-BPDE-N(2)-dG lesion led to a significant reduction in both the binding of UvrA and the incision efficiency of UvrABC by a factor of 5. These data suggest that E. coli NER features a dynamic two-stage recognition mechanism.     

Reduced sulfhydryls maintain specific incision of BPDE-DNA adducts by recombinant thermoresistant Bacillus caldotenax UvrABC endonuclease

Prokaryotic DNA repair nucleases are useful reagents for detecting DNA lesions. Escherichia coli UvrABC endonuclease can incise DNA containing UV photoproducts and bulky chemical adducts. The limited stability of the E. coli UvrABC subunits leads to difficulty in estimating incision efficiency and quantitative adduct detection. To develop a more stable enzyme with greater utility for the detection of DNA adducts, thermoresistant UvrABC endonuclease was cloned from the eubacterium Bacillus caldotenax (Bca) and individual recombinant protein subunits were overexpressed in and purified from E. coli. Here, we show that Bca UvrC that had lost activity or specificity could be restored by dialysis against buffer containing 500 mM KCl and 20mM dithiothreitol. Our data indicate that UvrC solubility depended on high salt concentrations and UvrC nuclease activity and the specificity of incisions depended on the presence of reduced sulfhydryls. Optimal conditions for BCA UvrABC-specific cleavage of plasmid DNAs treated with [3H](+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) (1-5 lesions/plasmid) were developed. Preincubation of substrates with UvrA and UvrB enhanced incision efficiency on damaged substrates and decreased non-specific nuclease activity on undamaged substrates. Under optimal conditions for damaged plasmid incision, approximately 70% of adducts were incised in 1 nM plasmid DNA (2 BPDE adducts/5.4 kbp plasmid) with UvrA at 2.5 nM, UvrB at 62.5 nM, and UvrC at 25 nM. These results demonstrate the potential usefulness of the Bca UvrABC for monitoring the distribution of chemical carcinogen-induced lesions in DNA.     

Malondialdehyde,a product of lipid peroxidation,is mutagenic in human cells

Malondialdehyde (MDA) is an endogenous genotoxic product of enzymatic and oxygen radical-induced lipid peroxidation whose adducts are known to exist in DNA isolated from healthy human beings. To evaluate the mutagenic potential of MDA in human cells, we reacted MDA with pSP189 shuttle vector DNA and then transfected them into human fibroblasts for replication. MDA induced up to a 15-fold increase in mutation frequency in the supF reporter gene compared with untreated DNA. Sequence analysis revealed that the majority of MDA-induced mutations occurred at GC base pairs. The most frequent mutations were large insertions and deletions, but base pair substitutions were also detected. MDA-induced mutations were completely abolished when the adducted shuttle vector was replicated in cells lacking nucleotide excision repair. MDA induction of large deletions and the apparent requirement for nucleotide excision repair suggested the possible involvement of a DNA interstrand cross-link as a premutagenic lesion. Indeed, MDA formed interstrand cross-links in duplex plasmids and oligonucleotides. Substrates containing the sequence 5'-d(CG) were preferentially cross-linked, consistent with the observation of base pair substitutions in 5'-d(CG) sites in the MDA-induced mutation spectrum. These experiments provide biological and biochemical evidence for the existence of MDA-induced DNA interstrand cross-links that could result from endogenous oxidative stress and likely have potent biological effects.     

Intrastrand DNA cross-links as tools for studying DNA replication and repair: two-, three-, and four-carbon tethers between the N(2) positions of adjacent guanines

A general protocol for preparation of oligonucleotides containing intrastrand cross-links between the exocyclic amino groups of adjacent deoxyguanosines has been developed. A series of 2, 3, and 4 methylene cross-links was incorporated site-specifically into an 11-mer (5'-GGCAGGTGGTG-3', cross-linked positions are underlined) via a reaction between oligonucleotide containing 2-fluoro-O(6)-trimethylsilylethyl deoxyinosines and the appropriate diamine (ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane). These cross-linked-oligonucleotides were studied for their ability to bend DNA by the method of Koo and Crothers [Koo, H. S., and Crothers, D. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1763-1767] in which the mobility of ligated oligomers in nondenaturing polyacrylamide gels is evaluated. It was found that all cross-links induced bending (2-carbon cross-link, 30.0 +/- 4.0 deg/turn; 3-carbon cross-link, 11.7 +/- 1.6 deg/turn; 4-carbon cross-link, 7.4 +/- 1.0 deg/turn). Despite the differing extent of helical distortion exhibited by the cross-links, all appeared to be equally blocking to replication by the Escherichia coli polymerases, pol I, pol II, and pol III. In contrast, when incision of the cross-links by the E. coli UvrABC nucleotide incision complex was studied, the extent of incision of the cross-link was found to correlate closely with the degree of bending measured in the gel mobility assay, i.e., the efficiency of incision was 2-carbon > 3-carbon > 4-carbon.     

The relative contribution of adduct blockage and DNA repair on template utilization during replication of 1,N2-propanodeoxyguanosine and pyrimido

The role of replication blockage by the exocyclic DNA adducts propanodeoxyguanosine (PdG) and pyrimido[1,2-alpha]purin-10(3H)-one (M1G) was determined through the use of site-specifically adducted M13MB102 genomes containing a C:C-mismatch approximately 3000 base-pairs from the site of adduct incorporation. Genomes containing either dG, PdG, or M1G positioned at site 6256 of the (-)-strand were transformed into repair-proficient and repair-deficient Escherichia coli strains and the percent template utilization was determined by hybridization analysis. Unmodified genomes containing a C:C-mismatch resulted in a percent template utilization of approximately 60 and 40% for the (-)- and (+)-strands, respectively. Transformation of PdG- or M(1)G-adducted genomes resulted in approximately a 60-40% and 50-50% (-)-strand to (+)-strand ratio, respectively, indicating that PdG and M(1)G are negligible blocks to replication in repair-proficient E. coli. This is in contrast to previous studies using (PdG:T)- and (M1G:T)-mismatched M13MB102 genomes, which resulted in a majority of the replication events using the unadducted (+)-strand and suggested that both adducts were significant blocks to replication [J. Biol. Chem. 272 (1997) 11434; Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 8652]. The C:C-mismatch results, though, indicate that the large strand bias detected in the earlier studies is due to repair of the adducts and resynthesis of the (-)-strand using the (+)-strand as a template for repair synthesis. Transformation of adducted C:C-mismatched genomes into E. coli strains deficient in nucleotide excision repair did result in an increased strand bias with only approximately 20 and 34% of the replication events using the (-)-strand for PdG- and M1G-adducted genomes, respectively. The increased strand bias indicates the importance of nucleotide excision repair in the removal of PdG and M1G.     

(5'S)-8,5'-cyclo-2'-deoxyguanosine is a strong block to replication, a potent pol V-dependent mutagenic lesion, and is inefficiently repaired in Escherichia coli

8,5'-Cyclopurines, making up an important class of ionizing radiation-induced tandem DNA damage, are repaired only by nucleotide excision repair (NER). They accumulate in NER-impaired cells, as in Cockayne syndrome group B and certain Xeroderma Pigmentosum patients. A plasmid containing (5'S)-8,5'-cyclo-2'-deoxyguanosine (S-cdG) was replicated in Escherichia coli with specific DNA polymerase knockouts. Viability was <1% in the wild-type strain, which increased to 5.5% with SOS. Viability decreased further in a pol II(-) strain, whereas it increased considerably in a pol IV(-) strain. Remarkably, no progeny was recovered from a pol V(-) strain, indicating that pol V is absolutely required for bypassing S-cdG. Progeny analyses indicated that S-cdG is significantly mutagenic, inducing ~34% mutation with SOS. Most mutations were S-cdG → A mutations, though S-cdG → T mutation and deletion of 5'C also occurred. Incisions of purified UvrABC nuclease on S-cdG, S-cdA, and C8-dG-AP on a duplex 51-mer showed that the incision rates are C8-dG-AP > S-cdA > S-cdG. In summary, S-cdG is a major block to DNA replication, highly mutagenic, and repaired slowly in E. coli.     

Excision repair of adozelesin-N3 adenine adduct by 3-methyladenine-DNA glycosylases and UvrABC nuclease

Adozelesin is a synthetic analog of the antitumor antibiotic CC-1065, which alkylates the N3 of adenine in the minor groove in a sequence-selective manner. Since the cytotoxic potency of a DNA alkylating agent can be modulated by DNA excision repair system, we investigated whether nucleotide excision repair (NER) and base excision repair (BER) enzymes are able to excise the bulky DNA adduct induced by adozelesin. The UvrABC nuclease and 3-methyladenine-DNA glycosylase, that exhibit a broad spectrum of substrate specificity, were selected as typical NER and BER enzymes, respectively. The adozelesin-DNA adduct was first formed in the radiolabeled restriction DNA fragment and its excision by purified repair enzymes was monitored on a DNA sequencing gel. The treatment of the DNA adduct with a purified UvrABC nuclease and sequencing gel analysis of cleaved DNA showed that UvrABC nuclease was able to incise the adozelesin adduct. The incision site corresponded to the general nuclease incision site. Excision of this adduct by 3-methyladenine-DNA glycosylases was determined following the treatment of the DNA adduct with a homogeneous recombinant bacterial, rat and human 3-methyladenine-DNA glycosylases. Abasic sites generated by DNA glycosyalses were cleaved by the associated lyase activity of the E. coli formamidopyrimidine-DNA glycosylase (Fpg). Resolution of cleaved DNA on a sequencing gel showed that the DNA glycosylase from different sources could not release the N3-adenine adducts. A cytotoxicity assay using E. coli repair mutant strains showed that E. coli mutant strains defective in the uvrA gene were more sensitive to cell killing by adozelesin than E. coli mutant strain defective in the alkA gene or the wild type. These results suggest that the NER pathway seems to be the major excision repair system in protecting cells from the cytotoxicity of adozelesin.     

Mutations predetermined by the primary structure of DNA

This study is concerned with an experimental verification of hypotheses postulating the involvement of self-complementary nucleotide sequences in the formation of deletions and insertions. It was suggested that deletions can arise in the regions of self-complementary nucleotide sequences, which allows the formation of the hairpin structures in a single-stranded DNA, arising during excision repair. These hairpin structures can be eliminated by nucleases or during DNA replication. Insertions can arise as a result of homologous recombination, when a migrating DNA strand contains a self-complementary sequence which forms hairpin structure. Model experiments were carried out with the pBR322 plasmid. A plasmid DNA with premutational damage in the palindrome-containing region was constructed by in vitro dimethylsulfate modification of one strand of EcoRI-BamHI restriction fragment. The plasmid was used for transformation of Escherichia coli. Restriction mapping and nucleotide analysis of the mutant DNAs demonstrated that they all contained deletions. The end points of the deletions coincide with the palindrome. To model homologous recombination, a plasmid with D-loop was constructed. A single-stranded DNA fragment containing palindrome forming a hairpin structure was introduced into the plasmid DNA and covalently fixed in the complex. When E. coli cells were transfected with this DNA, plasmid mutants containing insertions predetermined by palindromic structure arose. The evolutionary role of mutations predetermined by primary DNA structure is discussed.     

UvrABC nuclease complex repairs thymine glycol, an oxidative DNA base damage

The UvrABC nuclease complex recognizes a wide spectrum of DNA lesions including pyrimidine dimers, bulky chemical adducts and O6-methylguanine. In this study we have demonstrated that the UvrABC complex is also able to incise PM2 DNA containing the oxidative DNA lesion, thymine glycol. However, DNA containing dihydrothymine, a lesion with a similar structure to thymine glycol, was not incised. The UvrABC complex was also able to incise DNA containing reduced apurinic sites or apurinic sites modified with O-alkyl hydroxylamines, but not DNA containing apurinic sites or urea residues. In vivo, in the absence of base-excision repair, nucleotide excision repair was operable on phi X-174 RF transfecting DNA containing thymine glycols. The level of the repair was found to be directly related to the level of the UvrABC complex. Thus, UvrABC-mediated nucleotide excision repair appears to play a role in the repair of thymine glycol, an oxidative DNA-base lesion that is produced by ionizing radiation or formed during oxidative respiration.     

Initiation of repair of DNA-polypeptide cross-links by the UvrABC nuclease

Although the biochemical pathways that repair DNA-protein cross-links have not been clearly elucidated, it has been proposed that the partial proteolysis of cross-linked proteins into smaller oligopeptides constitutes an initial step in removal of these lesions by nucleotide excision repair (NER). To test the validity of this repair model, several site-specific DNA-peptide and DNA-protein cross-links were engineered via linkage at (1) an acrolein-derived gamma-hydroxypropanodeoxyguanosine adduct and (2) an apurinic/apyrimidinic site, and the initiation of repair was examined in vitro using recombinant proteins UvrA and UvrB from Bacillus caldotenax and UvrC from Thermotoga maritima. The polypeptides cross-linked to DNA were Lys-Trp-Lys-Lys, Lys-Phe-His-Glu-Lys-His-His-Ser-His-Arg-Gly-Tyr, and the 16 kDa protein, T4 pyrimidine dimer glycosylase/apurinic/apyrimidinic site lyase. For the substrates examined, DNA incision required the coordinated action of all three proteins and occurred at the eighth phosphodiester bond 5' to the lesion. The incision rates for DNA-peptide cross-links were comparable to or greater than that measured on fluorescein-adducted DNA, an excellent substrate for UvrABC. Incision rates were dependent on both the site of covalent attachment on the DNA and the size of the bound peptide. Importantly, incision of a DNA-protein cross-link occurred at a rate approximately 3.5-8-fold slower than the rates observed for DNA-peptide cross-links. Thus, direct evidence has been obtained indicating that (1) DNA-peptide cross-links can be efficiently incised by the NER proteins and (2) DNA-peptide cross-links are preferable substrates for this system relative to DNA-protein cross-links. These data suggest that proteolytic degradation of DNA-protein cross-links may be an important processing step in facilitating NER.     

Mutagenic frequencies of site-specifically located O6-methylguanine in wild-type Escherichia coli and in a strain deficient in ada-methyltransferase.

The adaptive response of Escherichia coli involves protection of the cells against the toxic and mutagenic consequences of exposure to high doses of a methylating agent by prior exposure to low doses of the agent. Ada protein, a major repair activity for O6-methylguanine, is activated to positively control the adaptive response; O6-methylguanine is one of the major mutagenic lesions produced by methylating agents. We investigated the mutation frequency of wild-type Escherichia coli and strains containing the ada-5 mutation in response to site-specifically synthesized O6-methylguanine under conditions in which the adaptive response was not induced. Site-directed mutagenesis and oligonucleotide self-selection techniques were used to isolate the progeny of M13mp18 DNAs constructed to contain O6-methylguanine at any of eight different positions. The progeny were isolated from E. coli strains isogeneic except for deficiency in Ada-methyltransferase repair, UvrABC excision repair, or both. The resulting O6-methylguanine mutation levels at each position were determined by using differential oligonucleotide hybridization. We found that the wild type had up to a 2.6-fold higher mutation frequency than ada-5 mutants. In addition, the mutation frequency varied with the position of the O6-methylguanine in the DNA in the wild type but not in ada-5 mutants; O6-methylguanine lesions at the 5' ends of runs of consecutive guanines gave the highest mutation frequencies. Determination of the mutation frequency of O6-methylguanine in wild-type and mutS cells showed that mismatch repair can affect O6-methylguanine mutation levels.     

RecA-mediated excision repair: a novel mechanism for repairing DNA lesions at sites of arrested DNA synthesis

In Escherichia coli, bulky DNA lesions are repaired primarily by nucleotide excision repair (NER). Unrepaired lesions encountered by DNA polymerase at the replication fork create a blockage which may be relieved through RecF-dependent recombination. We have designed an assay to monitor the different mechanisms through which a DNA polymerase blocked by a single AAF lesion may be rescued by homologous double-stranded DNA sequences. Monomodified single-stranded plasmids exhibit low survival in non-SOS induced E. coli cells; we show here that the presence of a homologous sequence enhances the survival of the damaged plasmid more than 10-fold in a RecA-dependent way. Remarkably, in an NER proficient strain, 80% of the surviving colonies result from the UvrA-dependent repair of the AAF lesion in a mechanism absolutely requiring RecA and RecF activity, while the remaining 20% of the surviving colonies result from homologous recombination mechanisms. These results uncover a novel mechanism - RecA-mediated excision repair - in which RecA-dependent pairing of the mono-modified single-stranded template with a complementary sequence allows its repair by the UvrABC excinuclease.     

Recombinational and mutagenic repair of psoralen interstrand cross-links in Saccharomyces cerevisiae

Psoralen photoreacts with DNA to form interstrand cross-links, which can be repaired by both nonmutagenic nucleotide excision repair and recombinational repair pathways and by mutagenic pathways. In the yeast Saccharomyces cerevisiae, psoralen cross-links are processed by nucleotide excision repair to form double-strand breaks (DSBs). In yeast, DSBs are repaired primarily by homologous recombination, predicting that cross-link and DSB repair should induce similar recombination end points. We compared psoralen cross-link, psoralen monoadduct, and DSB repair using plasmid substrates with site-specific lesions and measured the patterns of gene conversion, crossing over, and targeted mutation. Psoralen cross-links induced both recombination and mutations, whereas DSBs induced only recombination, and monoadducts were neither recombinogenic nor mutagenic. Although the cross-link- and DSB-induced patterns of plasmid integration and gene conversion were similar in most respects, they showed opposite asymmetries in their unidirectional conversion tracts: primarily upstream from the damage site for cross-links but downstream for DSBs. Cross-links induced targeted mutations in 5% of the repaired plasmids; all were base substitutions, primarily T --> C transitions. The major pathway of psoralen cross-link repair in yeast is error-free and involves the formation of DSB intermediates followed by homologous recombination. A fraction of the cross-links enter an error-prone pathway, resulting in mutations at the damage site.     

Binding discrimination of MutS to a set of lesions and compound lesions (base damage and mismatch) reveals its potential role as a cisplatin-damaged DNA sensing protein

The DNA mismatch repair (MMR) system plays a critical role in sensitizing both prokaryotic and eukaryotic cells to the clinically potent anticancer drug cisplatin. It is thought to mediate cytotoxicity through recognition of cisplatin DNA lesions. This drug generates a range of lesions that may also give rise to compound lesions resulting from the misincorporation of a base during translesion synthesis. Using gel mobility shift competition assays and surface plasmon resonance, we have analyzed the interaction of Escherichia coli MutS protein with site-specifically modified DNA oligonucleotides containing each of the four cisplatin cross-links or a set of compound lesions. The major 1,2-d(GpG) cisplatin intrastrand cross-link was recognized with only a 1.5-fold specificity, whereas a 47-fold specificity was found with a natural G/T containing DNA substrate. The rate of association, kon, for binding to the 1,2-d(GpG) adduct was 3.1 x 104 m-1 s-1 and the specificity of binding was essentially dependent on koff. DNA duplexes containing a single 1,2-d(ApG), 1,3-d(GpCpG) adduct, and an interstrand cross-link of cisplatin were not preferentially recognized. Among 12 DNA substrates, each containing a different cisplatin compound lesion derived from replicative misincorporation of one base opposite either of the 1,2-intrastrand adducts, 10 were specifically recognized including those that are more likely formed in vivo based on cisplatin mutation spectra. Moreover, among these lesions, two compound lesions formed when an adenine was misincorporated opposite a 1,2-d(GpG) adduct were not substrates for the MutY-dependent mismatch repair pathway. The ability of MutS to sense differentially various platinated DNA substrates suggests that cisplatin compound lesions formed during misincorporation of a base opposite either adducted base of both 1,2-intrastrand cross-links are more plausible critical lesions for MMR-mediated cisplatin cytotoxicity.     

Conformation, recognition by high mobility group domain proteins, and nucleotide excision repair of DNA intrastrand cross-links of novel antitumor trinuclear platinum complex BBR3464

The new antitumor trinuclear platinum compound [(trans-PtCl(NH(3))(2))(2)mu-trans-Pt(NH(3))(2)(H(2)N(CH(2))(6)NH(2))(2)](4+) (designated as BBR3464) is currently in phase II clinical trials. DNA is generally considered the major pharmacological target of platinum drugs. As such it is of considerable interest to understand the patterns of DNA damage. The bifunctional DNA binding of BBR3464 is characterized by the rapid formation of long range intra- and interstrand cross-links. We examined how the structures of the various types of the intrastrand cross-links of BBR3464 affect conformational properties of DNA, and how these adducts are recognized by high mobility group 1 protein and removed from DNA during in vitro nucleotide excision repair reactions. The results have revealed that intrastrand cross-links of BBR3464 create a local conformational distortion, but none of these cross-links results in a stable curvature. In addition, we have observed no recognition of these cross-links by high mobility group 1 proteins, but we have observed effective removal of these adducts from DNA by nucleotide excision repair. These results suggest that the processing of the intrastrand cross-links of BBR3464 in tumor cells sensitive to this drug may not be relevant to its antitumor effects. Hence, polynuclear platinum compounds apparently represent a novel class of platinum anticancer drugs acting by a different mechanism than cisplatin and its analogues.