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.     

Error-prone Translesion Synthesis Past DNA-Peptide Cross-links Conjugated to the Major Groove of DNA via C5 of Thymidine

DNA-protein cross-links (DPCs) are exceptionally bulky, structurally diverse DNA adducts formed in cells upon exposure to endogenous and exogenous bis-electrophiles, reactive oxygen species, and ionizing radiation. If not repaired, DPCs can induce toxicity and mutations. It has been proposed that the protein component of a DPC is proteolytically degraded, giving rise to smaller DNA-peptide conjugates, which can be subject to nucleotide excision repair and replication bypass. In this study, polymerase bypass of model DNA-peptide conjugates structurally analogous to the lesions induced by reactive oxygen species and DNA methyltransferase inhibitors was examined. DNA oligomers containing site-specific DNA-peptide conjugates were generated by copper-catalyzed [3 + 2] Huisgen cyclo-addition between an alkyne-functionalized C5-thymidine in DNA and an azide-containing 10-mer peptide. The resulting DNA-peptide conjugates were subjected to steady-state kinetic experiments in the presence of recombinant human lesion bypass polymerases κ and η, followed by PAGE-based assays to determine the catalytic efficiency and the misinsertion frequency opposite the lesion. We found that human polymerase κ and η can incorporate A, G, C, or T opposite the C5-dT-conjugated DNA-peptide conjugates, whereas human polymerase η preferentially inserts G opposite the lesion. Furthermore, HPLC-ESI⁻-MS/MS sequencing of the extension products has revealed that post-lesion synthesis was highly error-prone, resulting in mutations opposite the adducted site or at the +1 position from the adduct and multiple deletions. Collectively, our results indicate that replication bypass of peptides conjugated to the C5 position of thymine by human translesion synthesis polymerases leads to large numbers of base substitution and frameshift mutations.     

The modulation of topoisomerase I-mediated DNA cleavage and the induction of DNA-topoisomerase I crosslinks by crotonaldehyde-derived DNA adducts

Crotonaldehyde is a representative alpha,beta-unsaturated aldehyde endowed of mutagenic and carcinogenic properties related to its propensity to react with DNA. Cyclic crotonaldehyde-derived deoxyguanosine (CrA-PdG) adducts can undergo ring opening in duplex DNA to yield a highly reactive aldehydic moiety. Here, we demonstrate that site-specifically modified DNA oligonucleotides containing a single CrA-PdG adduct can form crosslinks with topoisomerase I (Top1), both directly and indirectly. Direct covalent complex formation between the CrA-PdG adduct and Top1 is detectable after reduction with sodium cyanoborohydride, which is consistent with the formation of a Schiff base between Top1 and the ring open aldehyde form of the adduct. In addition, we show that the CrA-PdG adduct alters the cleavage and religation activities of Top1. It suppresses Top1 cleavage complexes at the adduct site and induces both reversible and irreversible cleavage complexes adjacent to the CrA-PdG adduct. The formation of stable DNA-Top1 crosslinks and the induction of Top1 cleavage complexes by CrA-PdG are mutually exclusive. Lastly, we found that crotonaldehyde induces the formation of DNA-Top1 complexes in mammalian cells, which suggests a potential relationship between formation of DNA-Top1 crosslinks and the mutagenic and carcinogenic properties of crotonaldehyde.     

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.     

Repair of 8-methoxypsoralen monoadducts in mouse lymphoma cells

Studies of the repair of DNA lesions at biologically important doses is extremely difficult for most mutagens. With 8-methoxypsoralen (8-MOP) plus longwave ultraviolet light (UVA) as the lesion-inducing agent, however, it is easy to manipulate the relative frequency of different DNA adducts by means of a special experimental protocol (the tap-and-test protocol) and this can be used to measure repair of DNA adducts. Three classes of photoadducts are produced by 8-MOP plus UVA treatment: 3,4-cyclobutane monoadducts, 4',5'-cyclobutane monoadducts, and 8-MOP-DNA interstrand crosslinks. A monoadduct is formed when a photoactivated 8-MOP molecule reacts with a pyrimidine base. An 8-MOP-DNA interstrand crosslink is formed when an existing monoadduct is photoactivated to react with another pyrimidine base on the opposite DNA strand. Thus monoadducts are formed by absorption of one photon of light and crosslinks by absorption of two. In the tap-and-test experiments, cells were exposed to UVA in the presence of 8-MOP and then re-exposed to UVA in the absence of free 8-MOP so that only crosslinks can be produced by the second UVA treatment. By means of this technique we have previously shown that DNA crosslinks are much more effective than monoadducts at producing chromosomal damage (sister-chromatid exchanges and micronuclei) but not mutations (Liu-Lee et al., 1984). If L5178Y mouse lymphoma cells were able to remove monoadducts, incubation prior to the second UVA treatment should lead to decreases in the effect of re-irradiation, because fewer monoadducts would be available for crosslink formation. In this way, we have found that psoralen monoadducts are repaired in these cells and that about 70% of those capable of crosslink formation are removed or otherwise made unavailable for crosslink formation in 6 h.     

Site-specific mutagenesis by triple helix-forming oligonucleotides containing a reactive nucleoside analog

The specific recognition of homopurine–homo pyrimidine regions in duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation. Alkylation of nucleobases with functionalized TFOs would have the potential for site-directed mutagenesis. Recently, we demonstrated that a TFO bearing 2-amino-6-vinylpurine derivative, 1, achieves triplex-mediated reaction with high selectivity toward the cytosine of the G-C target site. In this report, we have investigated the use of this reagent to target mutations to a specific site in a shuttle vector plasmid, which replicates in mammalian cells. TFOs bearing 1 produced adducts at the complementary position of 1 and thereby introduced mutations at that site during replication/repair of the plasmid in mammalian cells. Reagents that produce covalent cytosine modifications are relatively rare. These TFOs enable the preparation of templates carrying targeted cytosine adducts for in vitro and in vivo studies. The ability to target mutations may prove useful as a tool for studying DNA repair, and as a technique for gene therapy and genetic engineering.     

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.     

1,N2-deoxyguanosine adducts of acrolein,crotonaldehyde, and trans-4-hydroxynonenal cross-link to peptides via Schiff base linkage

DNA-protein cross-links (DPCs) are formed upon exposure to a variety of chemical and physical agents and pose a threat to genomic integrity. In particular, acrolein and related aldehydes produce DPCs, although the chemical linkages for such cross-links have not been identified. Here, we report that oligodeoxynucleotides containing 1,N(2)-deoxyguanosine adducts of acrolein, crotonaldehyde, and trans-4-hydroxynonenal can form cross-links with the tetrapeptide Lys-Trp-Lys-Lys. We concluded that complex formation is mediated by a Schiff base linkage because DNA-peptide complexes were covalently trapped following reduction with sodium cyanoborohydride, and pre-reduction of adducted DNAs inhibited complex formation. A previous NMR study demonstrated that duplex DNA catalyzes ring opening for the acrolein-derived gamma-hydroxy-1,N(2)-propanodeoxyguanosine adduct to yield an aldehydic function (de los Santos, C., Zaliznyak, T., and Johnson, F. (2001) J. Biol. Chem. 276, 9077-9082). Consistent with this earlier observation, the adducts under investigation were more reactive in duplex DNA than in single-stranded DNA, and we concluded that the ring-open aldehydic moiety is the induced tautomer in duplex DNA for adducts exhibiting high relative reactivity. Adducted DNA cross-linked to Arg-Trp-Arg-Arg and Lys-Trp-Lys-Lys with comparable efficiency, and N(alpha)-acetylation of peptides dramatically inhibited trapping; thus, the reactive nucleophile is located at the N-terminal alpha-amine of the peptide. These data suggest that Schiff base chemistry can mediate DPC formation in vivo following the formation of stable aldehyde-derived DNA adducts.     

Mutagenicity, cytotoxicity and DNA crosslinking in V79 Chinese hamster cells treated with cis- and trans-Pt(II) diamminedichloride.

The mutagenicity and cytotoxicity of cis- and trans-Pt(II) diamminedichloride (PDD) were examined in V79 Chinese hamster lung cells and compared with effects on DNA measured by alkaline elution. DNA--protein crosslinks and DNA interstrand crosslinks were detected following doses of cis-PDD which reduced cell survival 80--90% and which produced a mutant frequency of 3 X 10(-4) at the HGPRT locus. Equitoxic doses of trans-PDD were much less mutagenic than cis-PDD. At equitoxic doses, trans-PDD produced more DNA-protein crosslinking than did cis-PDD, but interstrand crosslinking for the two isomers was comparable. Hence, the interstrand crosslink could be the cytotoxic lesion produced by these Pt compounds whereas neither of these DNA lesions are necessarily mutagenic. The mutagenesis produced by cis-PDD could be due to crosslinks of a different type than those produced by trans-PDD or it may be due to monofunctional damage.     

A cell cycle-associated pathway for repair of DNA-protein crosslinks in mammalian cells

Bulky adducts to DNA including DNA-protein crosslinks formed with trans-platinum(II)diammine-dichloride are repaired largely by the nucleotide excision pathway in mammalian cells. The discovery in this laboratory that cells deficient in nucleotide excision repair, i.e., SV40-virus transformed SV-XP20S cells, can efficiently repair DNA-protein crosslinks implicates a second pathway. In this report, details concerning this pathway are presented. DNA-protein crosslinks induced with 20 microM trans-platinum were assayed by the membrane alkaline elution procedure of Kohn. DNA replication was measured by CsCl gradient separation of newly synthesized DNA that had incorporated 5-bromodeoxyuridine. The following results indicate that this new repair pathway is associated with cell cycling: Whereas rapidly proliferating human cells deficient in excision repair (SV40 transformed XP20S, group A) are proficient in repair of DNA-protein crosslinks, the more slowly growing untransformed parent line is deficient but can complete repair after prolonged periods of 4-6 days, the approximate doubling time of the cell population. Either "used" culture medium or cycloheximide (1 microgram/ml) inhibits cell proliferation, protein synthesis, DNA replication and crosslink repair. In the presence of increasing concentrations of cycloheximide (0.01-5 micrograms/ml) the percent of DNA replication decreases and is essentially equivalent to the percent of crosslink repair. The following results indicate that this new repair pathway, though associated with cell cycling, is independent of DNA replication per se. The rates of DNA-protein crosslink repair and DNA replication are essentially the same in mouse L1210 cells rapidly proliferating in 20% serum supplement; however, to slower proliferation rates in 1% serum rate of crosslink repair is slower but differs from that of DNA replication. In the presence of aphidicolin (10 micrograms/ml) cells can repair DNA-protein crosslinks in virtually the complete absence of DNA replication, though the rate is slower in both nucleotide excision-proficient and -deficient cells. Thus, DNA replication is not essential for repair of DNA-protein crosslinks. Comparison of the kinetics of replication and DNA-protein crosslink repair of pulse-labeled indicates that, in the absence of metabolic inhibitors, repair of the crosslinks is independent of replication per se and, therefore, DNA recombination events are not involved in this repair process. We conclude, therefore, that the new repair pathway is not coupled with DNA replication but is with cell cycling.     

A novel function of DNA polymerase zeta regulated by PCNA

DNA polymerase zeta (Polzeta) participates in translesion DNA synthesis and is involved in the generation of the majority of mutations induced by DNA damage. The mechanisms that license access of Polzeta to the primer terminus and regulate the extent of its participation in genome replication are poorly understood. The Polzeta-dependent damage-induced mutagenesis requires monoubiquitination of proliferating cell nuclear antigen (PCNA) that is triggered by exposure to mutagens. We show that Polzeta contributes to DNA replication and causes mutagenesis not only in response to DNA damage but also in response to malfunction of normal replicative machinery due to mutations in replication genes. These replication defects lead to ubiquitination of PCNA even in the absence of DNA damage. Unlike damage-induced mutagenesis, the Polzeta-dependent spontaneous mutagenesis in replication mutants is reduced in strains defective in both ubiquitination and sumoylation of Lys164 of PCNA. Additionally, studies of a PCNA mutant defective for functional interactions with Polzeta, but not for monoubiquitination by the Rad6/Rad18 complex demonstrate a role for PCNA in regulating the mutagenic activity of Polzeta separate from its modification at Lys164.     

The Fanconi anaemia gene FANCC promotes homologous recombination and error-prone DNA repair

The Fanconi anemia (FA) protein FANCC is essential for chromosome stability in vertebrate cells, a feature underscored by the extreme sensitivity of FANCC-deficient cells to agents that crosslink DNA. However, it is not known how this FA protein facilitates the repair of both endogenously acquired and mutagen-induced DNA damage. Here, we use the model vertebrate cell line DT40 to address this question. We discover that apart from functioning in homologous recombination, FANCC also promotes the mutational repair of endogenously generated abasic sites. Moreover in these vertebrate cells, the efficient repair of crosslinks requires the combined functions of FANCC, translesion synthesis, and homologous recombination. These studies reveal that the FA proteins cooperate with key mutagenesis and repair processes that enable replication of damaged DNA.     

Cr(III)-mediated crosslinks of glutathione or amino acids to the DNA phosphate backbone are mutagenic in human cells.

Carcinogenic Cr(VI) compounds were previously found to induce amino acid/glutathione-Cr(III)-DNA crosslinks with the site of adduction on the phosphate backbone. Utilizing the pSP189 shuttle vector plasmid we found that these ternary DNA adducts were mutagenic in human fibroblasts. The Cr(III)-glutathione adduct was the most potent in this assay, followed by Cr(III)-His and Cr(III)-Cys adducts. Binary Cr(III)-DNA complexes were only weakly mutagenic, inducing a significant response only at a 10 times higher number of adducts compared with Cr(III)-glutathione. Single base substitutions at the G:C base pairs were the predominant type of mutations for all Cr(III) adducts. Cr(III), Cr(III)-Cys and Cr(III)-His adducts induced G:C-->A:T transitions and G:C-->T:A transversions with almost equal frequency, whereas the Cr(III)-glutathione mutational spectrum was dominated by G:C-->T:A transversions. Adduct-induced mutations were targeted toward G:C base pairs with either A or G in the 3' position to the mutated G, while spontaneous mutations occurred mostly at G:C base pairs with a 3' A. No correlation was found between the sites of DNA adduction and positions of base substitution, as adducts were formed randomly on DNA with no base specificity. The observed mutagenicity of Cr(III)-induced phosphotriesters demonstrates the importance of a Cr(III)-dependent pathway in Cr(VI) carcinogenicity.     

Mutagenic DNA repair in Escherichia coli. VII. Constitutive and inducible manifestations.

Incubation of E. coli WP2 in the presence of chloramphenicol (CAP) for 90 min before and 60 min after γ-irradiation had no effect on the induction of Trp⁺ mutations. Bacteria that had been treated with CAP for 90 min prior to UV irradiation showed normal or near normal yields of induced mutations to streptomycin or colicin E2 resistance. Most of these mutations lost their photoreversibility (indicating “fixation”) during continued incubation with CAP for a further 60 min after irradiation, during which time neither protein nor DNA synthesis was detectable. It is suggested that CAP-sensitive protein synthesis is not required for mutagenic (error-prone) repair of lesions in pre-existing DNA, arguing against an inducible component in this repair.In contrast the frequency of UV-induced mutations to Trp⁺ (largely at suppressor loci) was drastically reduced by CAP pretreatment, confirming the need for an active replication fork for UV-mutagenesis at these loci. It is known from the work of others that CAP given after UV abolishes mutagenesis at these loci. We conclude that CAP-sensitive protein synthesis (consistent with a requirement for an inducible function) is necessary for mutagenic repair only in newly-replicated DNA (presumably at daughter strand gaps) and not in pre-existing DNA. The data are consistent with but do not prove the hypothesis that CAP-sensitive and insensitive modes of mutagenesis reflect minor differences in the operation of a single basic mutagenic repair system.     

Adducts formed by the food mutagen 2-amino-3-methylimidazo(4, 5-f ) quinoline induce frameshift mutations at hot spots through an SOS-independent pathway

The potency of 2-amino-3-methylimidazo(4, 5-f)quinoline (IQ) adducts to induce ?2, ?1 and +1 frameshift mutations has been determined on specific target DNA sequences, namely short runs of alternating GpC sequences and short runs of guanines. The genetic control of the mutational processes has been analyzed using different Escherichia coli mutants, affected either in the control or in the mutagenesis pathway of the SOS system. We have shown that IQ adducts induce very efficiently both ?1 and ?2 frameshift mutations in E. coli. Both types of deletion mutations are induced in bacteria without the need of SOS induction, indicating that no LexA-controlled functions, in particular the UmuDC proteins, are required for mutation fixation. We have also shown that the frequency of IQ-induced ?2 frameshift mutations in alternating GC sequences increases with the length of the repetition. The efficiency of IQ adducts to induce ?1 and ?2 frameshift mutations is similar to that of N‐2-acetylaminofluorene (AAF) adducts. Both chemicals are potent carcinogens which form covalent adducts at the C8 position of guanines. We suggest that in both cases the adduct-induced DNA structure allows the replication complex to perform a mutagenic bypass of the lesion by a slippage mechanism. However, in contrast to AAF-induced frameshift mutagenesis, IQ-induced frameshift mutagenesis is SOS-independent.     

PCNA monoubiquitylation and DNA polymerase η ubiquitin-binding domain are required to prevent 8-oxoguanine-induced mutagenesis in Saccharomyces cerevisiae

7,8-Dihydro-8-oxoguanine (8-oxoG) is an abundant and mutagenic DNA lesion. In Saccharomyces cerevisiae, the 8-oxoG DNA N-glycosylase (Ogg1) acts as the primary defense against 8-oxoG. Here, we present evidence for cooperation between Rad18–Rad6-dependent monoubiquitylation of PCNA at K164, the damage-tolerant DNA polymerase η and the mismatch repair system (MMR) to prevent 8-oxoG-induced mutagenesis. Preventing PCNA modification at lysine 164 (pol30-K164R) results in a dramatic increase in GC to TA mutations due to endogenous 8-oxoG in Ogg1-deficient cells. In contrast, deletion of RAD5 or SIZ1 has little effect implying that the modification of PCNA relevant for preventing 8-oxoG-induced mutagenesis is monoubiquitin as opposed to polyubiquitin or SUMO. We also report that the ubiquitin-binding domain (UBZ) of Pol η is essential to prevent 8-oxoG-induced mutagenesis but only in conjunction with a functional PCNA-binding domain (PIP). We propose that PCNA is ubiquitylated during the repair synthesis reaction after the MMR-dependent excision of adenine incorporated opposite to 8-oxoG. Monoubiquitylation of PCNA would favor the recruitment of Pol η thereby allowing error-free incorporation of dCMP opposite to 8-oxoG. This study suggests that Pol η and the post-replication repair (PRR) machinery can also prevent mutagenesis at DNA lesions that do not stall replication forks.     

Detection of oxaliplatin-induced DNA crosslinks in vitro and in cancer patients using the alkaline comet assay

Oxaliplatin is frequently used in the therapy of cancer. In DNA, oxaliplatin induces, like cisplatin, the formation of crosslinks, which are commonly accepted as being responsible for the cytotoxicity of platinum agents. The detection of oxaliplatin-induced DNA crosslink formation and repair could be a good measure of assessing how a patient is responding to the agent. In this study, we used a validated modification of the alkaline comet assay for detecting the presence of these crosslinks in vitro and in cancer patients. The H460 tumour cell line was treated in vitro with a range of oxaliplatin and cisplatin doses, and the subsequent crosslink formation and repair compared between the two agents. In addition, lymphocytes from cancer patients undergoing oxaliplatin-based chemotherapy were assayed for the formation and repair of oxaliplatin-induced crosslinks. A dose-response was observed in the in vitro samples, with cisplatin producing more crosslinks than oxaliplatin at equimolar concentrations and lesions induced by both agents showing different repair efficiencies. Furthermore, evidence of crosslink formation and repair was observed in the peripheral blood lymphocytes of all cancer patients studied, along with the detection of interindividual variability in crosslink formation and repair efficiencies. To the best of our knowledge, this is the first time that oxaliplatin DNA crosslinks have been detected either in vitro or in patient samples using the alkaline comet assay. Due to its sensitivity, rapidity, small cell sample and low cost, the alkaline comet assay is a good method for the detection of oxaliplatin-induced crosslinks and their subsequent repair and, in future clinical studies, could prove to be a valuable tool in assessing/predicting a patient's response to chemotherapy.     

Formation of lysine 63-linked poly-ubiquitin chains protects human lung cells against benzo[a]pyrene-diol-epoxide-induced mutagenicity

Benzo[a]pyrene exerts its mutagenic effects via induction of benzo[a]pyrene-diol-epoxide (BPDE)-DNA adducts. Such helix-distorting adducts are not always successfully repaired prior to DNA replication, which may result in a blocked replication fork. To alleviate this stall, cells utilize DNA damage tolerance systems involving either error-free damage avoidance or error-prone translesion synthesis. Studies in yeast suggest the modification of PCNA by lysine 63-linked poly-ubiquitin (K63-polyUb) chains as a key mediator of the error-free damage avoidance pathway. Recently, we extended this observation to human cells, showing the occurrence of poly-ubiquitination of PCNA in UV-irradiated human cells. In the present study, we hypothesized that disrupting the formation of K63-polyUb chains inhibits damage avoidance and favors error-prone repair involving low-fidelity polymerases (e.g. POLeta), causing increased BPDE-induced mutagenicity. To test this hypothesis, we generated A549 cells expressing either a mutant ubiquitin (K63R-Ub) which blocks further ubiquitination through K63, or the wild type ubiquitin (WT-Ub). We show that PCNA is poly-ubiquitinated in these cells upon BPDE-exposure and that disruption of K63-polyUb chain formation has no effect on BPDE-induced toxicity. In contrast, significantly higher frequencies of BPDE-induced HPRT mutations were observed in K63R-Ub expressing cells, of which the majority (74%) was G-->T transversion. BPDE treatment caused an enhanced recruitment of POLeta to the replication machinery of the K63R-Ub expressing cells, where it co-localized with PCNA. Suppression of POLeta expression by using siRNA resulted in a 50% reduction of BPDE-induced mutations in the K63R cells. In conclusion, we demonstrated that formation of K63-polyUb chains protects BPDE-exposed human cells against translesion synthesis-mediated mutagenesis. These findings indicate that K63-polyubiquitination guards against chemical carcinogenesis by preventing mutagenesis and thus contributing to genomic stability.     

Genotoxicity and mutagenicity of chromium(VI)/ascorbate-generated DNA adducts in human and bacterial cells

Reduction of carcinogenic Cr(VI) by vitamin C generates ascorbate-Cr(III)-DNA cross-links, binary Cr(III)-DNA adducts, and can potentially cause oxidative DNA damage by intermediate reaction products. Here, we examined the mutational spectrum and the importance of different forms of DNA damage in genotoxicity and mutagenicity of Cr(VI) activated by physiological concentrations of ascorbate. Reduction of Cr(VI) led to a dose-dependent formation of both mutagenic and replication-blocking DNA lesions as detected by propagation of the pSP189 plasmids in human fibroblasts. Disruption of Cr-DNA binding abolished mutagenic responses and normalized the yield of replicated plasmids, indicating that Cr-DNA adducts were responsible for both mutagenicity and genotoxicity of Cr(VI). The absence of DNA breaks and abasic sites confirmed the lack of a significant production of hydroxyl radicals and Cr(V)-peroxo complexes in Cr(VI)-ascorbate reactions. Ascorbate-Cr(III)-DNA cross-links were much more mutagenic than smaller Cr(III)-DNA adducts and accounted for more than 90% of Cr(VI) mutagenicity. Ternary adducts were also several times more potent in the inhibition of replication than binary complexes. The Cr(VI)-induced mutational spectrum consisted of an approximately equal number of deletions and G/C-targeted point mutations (51% G/C --> T/A and 30% G/C --> A/T). In Escherichia coli cells, Cr(VI)-induced DNA adducts were only highly genotoxic but not mutagenic under either normal or SOS-induced conditions. Lower toxicity and high mutagenicity of ascorbate-Cr(III)-DNA adducts in human cells may result from the recruitment of an error-prone bypass DNA polymerase(s) to the stalled replication forks. Our results suggest that phosphotriester-type DNA adducts could play a more important role in human than bacterial mutagenesis.     

Rescue of replication failure by Fanconi anaemia proteins

Chromosomal aberrations are often associated with incomplete genome duplication, for instance at common fragile sites, or as a consequence of chemical alterations in the DNA template that block replication forks. Studies of the cancer-prone disease Fanconi anaemia (FA) have provided important insights into the resolution of replication problems. The repair of interstrand DNA crosslinks induced by chemotherapy drugs is coupled with DNA replication and controlled by FA proteins. We discuss here the recent discovery of new FA-associated proteins and the development of new tractable repair systems that have dramatically improved our understanding of crosslink repair. We focus also on how FA proteins protect against replication failure in the context of fragile sites and on the identification of reactive metabolites that account for the development of Fanconi anaemia symptoms.