Irina G. Minko

Chemistry and Biology of DNA Containing 1,N2-Deoxyguanosine Adducts of the α,β-Unsaturated Aldehydes Acrolein, Crotonaldehyde, and 4-Hydroxynonenal

The α,β-unsaturated aldehydes (enals) acrolein, crotonaldehyde, and trans-4-hydroxynonenal (4-HNE) are products of endogenous lipid peroxidation, arising as a consequence of oxidative stress. The addition of enals to dG involves Michael addition of the N2-amine to give N2-(3-oxopropyl)-dG adducts, followed by reversible cyclization of N1 with the aldehyde, yielding 1,N2-dG exocyclic products. The 1,N2-dG exocyclic adducts from acrolein, crotonaldehyde, and 4-HNE exist in human and rodent DNA. The enal-induced 1,N2-dG lesions are repaired by the nucleotide excision repair pathway in both Escherichia coli and mammalian cells. Oligodeoxynucleotides containing structurally defined 1,N2-dG adducts of acrolein, crotonaldehyde, and 4-HNE were synthesized via a postsynthetic modification strategy. Site-specific mutagenesis of enal adducts has been carried out in E. coli and various mammalian cells. In all cases, the predominant mutations observed are G→T transversions, but these adducts are not strongly miscoding. When placed into duplex DNA opposite dC, the 1,N2-dG exocyclic lesions undergo ring opening to the corresponding N2-(3-oxopropyl)-dG derivatives. Significantly, this places a reactive aldehyde in the minor groove of DNA, and the adducted base possesses a modestly perturbed Watson−Crick face. Replication bypass studies in vitro indicate that DNA synthesis past the ring-opened lesions can be catalyzed by pol η, pol ι, and pol κ. It also can be accomplished by a combination of Rev1 and pol ζ acting sequentially. However, efficient nucleotide insertion opposite the 1,N2-dG ring-closed adducts can be carried out only by pol ι and Rev1, two DNA polymerases that do not rely on the Watson−Crick pairing to recognize the template base. The N2-(3-oxopropyl)-dG adducts can undergo further chemistry, forming interstrand DNA cross-links in the 5′-CpG-3′ sequence, intrastrand DNA cross-links, or DNA−protein conjugates. NMR and mass spectrometric analyses indicate that the DNA interstand cross-links contain a mixture of carbinolamine and Schiff base, with the carbinolamine forms of the linkages predominating in duplex DNA. The reduced derivatives of the enal-mediated N2-dG:N2-dG interstrand cross-links can be processed in mammalian cells by a mechanism not requiring homologous recombination. Mutations are rarely generated during processing of these cross-links. In contrast, the reduced acrolein-mediated N2-dG peptide conjugates can be more mutagenic than the corresponding monoadduct. DNA polymerases of the DinB family, pol IV in E. coli and pol κ in human, are implicated in error-free bypass of model acrolein-mediated N2-dG secondary adducts, the interstrand cross-links, and the peptide conjugates.

Role for DNA Polymerase κ in the Processing of N2-N2-Guanine Interstrand Cross-links

Although there exists compelling genetic evidence for a homologous recombination-independent pathway for repair of interstrand cross-links (ICLs) involving translesion synthesis (TLS), biochemical support for this model is lacking. To identify DNA polymerases that may function in TLS past ICLs, oligodeoxynucleotides were synthesized containing site-specific ICLs in which the linkage was between N2-guanines, similar to cross-links formed by mitomycin C and enals. Here, data are presented that mammalian cell replication of DNAs containing these lesions was ∼97% accurate. Using a series of oligodeoxynucleotides that mimic potential intermediates in ICL repair, we demonstrate that human polymerase (pol) κ not only catalyzed accurate incorporation opposite the cross-linked guanine but also replicated beyond the lesion, thus providing the first biochemical evidence for TLS past an ICL. The efficiency of TLS was greatly enhanced by truncation of both the 5 ′ and 3 ′ ends of the nontemplating strand. Further analyses showed that although yeast Rev1 could incorporate a dCTP opposite the cross-linked guanine, no evidence was found for TLS by pol ζ or a pol ζ/Rev1 combination. Because pol κ was able to bypass these ICLs, biological evidence for a role for pol κ in tolerating the N2-N2-guanine ICLs was sought; both cell survival and chromosomal stability were adversely affected in pol κ-depleted cells following mitomycin C exposure. Thus, biochemical data and cellular studies both suggest a role for pol κ in the processing of N2-N2-guanine ICLs.

Efficient and Error-Free Replication Past a Minor-Groove DNA Adduct by the Sequential Action of Human DNA Polymerases ι and κ

ABSTRACT DNA polymerase ι (Polι) is a member of the Y family of DNA polymerases, which promote replication through DNA lesions. The role of Polι in lesion bypass, however, has remained unclear. Polι is highly unusual in that it incorporates nucleotides opposite different template bases with very different efficiencies and fidelities. Since interactions of DNA polymerases with the DNA minor groove provide for the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, we considered the possibility that Polι differs from other DNA polymerases in not being as sensitive to distortions of the minor groove at the site of the incipient base pair and that this enables it to incorporate nucleotides opposite highly distorting minor-groove DNA adducts. To check the validity of this idea, we examined whether Polι could incorporate nucleotides opposite the γ-HOPdG adduct, which is formed from an initial reaction of acrolein with the N2 of guanine. We show here that Polι incorporates a C opposite this adduct with nearly the same efficiency as it does opposite a nonadducted template G residue. The subsequent extension step, however, is performed by Polκ, which efficiently extends from the C incorporated opposite the adduct. Based upon these observations, we suggest that an important biological role of Polι and Polκ is to act sequentially to carry out the efficient and accurate bypass of highly distorting minor-groove DNA adducts of the purine bases.

Incision of DNA–protein crosslinks by UvrABC nuclease suggests a potential repair pathway involving nucleotide excision repair

DNA–protein crosslinks (DPCs) arise in biological systems as a result of exposure to a variety of chemical and physical agents, many of which are known or suspected carcinogens. The biochemical pathways for the recognition and repair of these lesions are not well understood in part because of methodological difficulties in creating site-specific DPCs. Here, a strategy for obtaining site-specific DPCs is presented, and in vitro interactions of the Escherichia coli nucleotide excision repair (NER) UvrABC nuclease at sites of DPCs are investigated. To create site-specific DPCs, the catalytic chemistry of the T4 pyrimidine dimer glycosylase/apurinic/apyrimidinic site lyase (T4-pdg) has been exploited, namely, its ability to be covalently trapped to apurinic/apyrimidinic sites within duplex DNA under reducing conditions. Incubation of the DPCs with UvrABC proteins resulted in DNA incision at the 8th phosphate 5′ and the 5th and 6th phosphates 3′ to the protein-adducted site, generating as a major product of the reaction a 12-mer DNA fragment crosslinked with the protein. The incision occurred only in the presence of all three protein subunits, and no incisions were observed in the nondamaged complementary strand. The UvrABC nuclease incises DPCs with a moderate efficiency. The proper assembly and catalytic function of the NER complex on DNA containing a covalently attached 16-kDa protein suggest that the NER pathway may be involved in DPC repair and that at least some subset of DPCs can be removed by this mechanism without prior proteolytic degradation.

Efficient and error-free replication past a minor-groove N2-guanine adduct by the sequential action of yeast Rev1 and DNA polymerase ζ

ABSTRACT Rev1, a member of the Y family of DNA polymerases, functions in lesion bypass together with DNA polymerase ζ (Polζ). Rev1 is a highly specialized enzyme in that it incorporates only a C opposite template G. While Rev1 plays an indispensable structural role in Polζ-dependent lesion bypass, the role of its DNA synthetic activity in lesion bypass has remained unclear. Since interactions of DNA polymerases with the DNA minor groove contribute to the nearly equivalent efficiencies and fidelities of nucleotide incorporation opposite each of the four template bases, here we examine the possibility that unlike other DNA polymerases, Rev1 does not come into close contact with the minor groove of the incipient base pair, and that enables it to incorporate a C opposite the N2-adducted guanines in DNA. To test this idea, we examined whether Rev1 could incorporate a C opposite the γ-hydroxy-1,N 2-propano-2′deoxyguanosine DNA minor-groove adduct, which is formed from the reaction of acrolein with the N 2 of guanine. Acrolein, an α,β-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from other oxidation reactions. We show here that Rev1 efficiently incorporates a C opposite this adduct from which Polζ subsequently extends, thereby completing the lesion bypass reaction. Based upon these observations, we suggest that an important role of the Rev1 DNA synthetic activity in lesion bypass is to incorporate a C opposite the various N 2-guanine DNA minor-groove adducts that form in DNA.

Replication Bypass of the Acrolein-Mediated Deoxyguanine DNA- Peptide Cross-links by DNA polymerases of the DinB Family

DNA-protein cross-links (adducts) are formed in cellular DNA under a variety of conditions, particularly following exposure to an alpha,beta-unsaturated aldehyde, acrolein. DNA-protein cross-links are subject to repair or damage-tolerance processes. These adducts serve as substrates for proteolytic degradation, yielding DNA-peptide lesions that have been shown to be actively repaired by the nucleotide excision repair complex. Alternatively, DNA-peptide cross-links can be subjected to replication bypass. We present new evidence about the capabilities of DNA polymerases to synthesize DNA past such cross-links. DNAs were constructed with site-specific cross-links, in which either a tetrapeptide or a dodecylpeptide was covalently attached at the N (2) position of guanine via an acrolein adduct, and replication bypass assays were carried out with members of the DinB family of polymerases, human polymerase (pol) kappa, Escherichia coli pol IV, and various E. coli polymerases that do not belong to the DinB family. Pol kappa was able to catalyze both the incorporation and the extension steps with an efficiency that was qualitatively indistinguishable from control (undamaged) substrates. Fidelity was comparable on all of these substrates, suggesting that pol kappa would have a role in the low mutation frequency associated with replication of these adducts in mammalian cells. When the E. coli orthologue of pol kappa, damage-inducible DNA polymerase, pol IV, was analyzed on the same substrates, pause sites were detected opposite and three nucleotides beyond the site of the lesion, with incorporation opposite the lesion being accurate. In contrast, neither E. coli replicative polymerase, pol III, nor E. coli damage-inducible polymerases, pol II and pol V, could efficiently incorporate a nucleotide opposite the DNA-peptide cross-links. Consistent with a role for pol IV in tolerance of these lesions, the replication efficiency of DNAs containing DNA-peptide cross-links was greatly reduced in pol IV-deficient cells. Collectively, these data indicate an important role for the DinB family of polymerases in tolerance mechanisms of N (2)-guanine-linked DNA-peptide cross-links.

Replication bypass of interstrand cross-link intermediates by Escherichia coli DNA polymerase IV.

Repair of interstrand DNA cross-links (ICLs) in Escherichia coli can occur through a combination of nucleotide excision repair (NER) and homologous recombination. However, an alternative mechanism has been proposed in which repair is initiated by NER followed by translesion DNA synthesis (TLS) and completed through another round of NER. Using site-specifically modified oligodeoxynucleotides that serve as a model for potential repair intermediates following incision by E. coli NER proteins, the ability of E. coli DNA polymerases (pol) II and IV to catalyze TLS past N2-N2-guanine ICLs was determined. No biochemical evidence was found suggesting that pol II could bypass these lesions. In contrast, pol IV could catalyze TLS when the nucleotides that are 5′ to the cross-link were removed. The efficiency of TLS was further increased when the nucleotides 3′ to the cross-linked site were also removed. The correct nucleotide, C, was preferentially incorporated opposite the lesion. When E. coli cells were transformed with a vector carrying a site-specific N2-N2-guanine ICL, the transformation efficiency of a pol II-deficient strain was indistinguishable from that of the wild type. However, the ability to replicate the modified vector DNA was nearly abolished in a pol IV-deficient strain. These data strongly suggest that pol IV is responsible for TLS past N2-N2-guanine ICLs.

Replication past a trans-4-Hydroxynonenal Minor-Groove Adduct by the Sequential Action of Human DNA Polymerases ι and κ

ABSTRACT The X-ray crystal structure of human DNA polymerase ι (Polι) has shown that it differs from all known Pols in its dependence upon Hoogsteen base pairing for synthesizing DNA. Hoogsteen base pairing provides an elegant mechanism for synthesizing DNA opposite minor-groove adducts that present a severe block to synthesis by replicative DNA polymerases. Germane to this problem, a variety of DNA adducts form at the N2 minor-groove position of guanine. Previously, we have shown that proficient and error-free replication through the γ-HOPdG (γ-hydroxy-1,N 2-propano-2′-deoxyguanosine) adduct, which is formed from the reaction of acrolein with the N2 of guanine, is mediated by the sequential action of human Polι and Polκ, in which Polι incorporates the nucleotide opposite the lesion site and Polκ carries out the subsequent extension reaction. To test the general applicability of these observations to other adducts formed at the N2 position of guanine, here we examine the proficiency of human Polι and Polκ to synthesize past stereoisomers of trans-4-hydroxy-2-nonenal-deoxyguanosine (HNE-dG). Even though HNE- and acrolein-modified dGs share common structural features, due to their increased size and other structural differences, HNE adducts are potentially more blocking for replication than γ-HOPdG. We show here that the sequential action of Polι and Polκ promotes efficient and error-free synthesis through the HNE-dG adducts, in which Polι incorporates the nucleotide opposite the lesion site and Polκ performs the extension reaction.

Novel Enzymatic Function of DNA Polymerase v in Translesion DNA Synthesis Past Major Groove DNA―Peptide and DNA―DNA Cross-Links

DNA polymerase ν (POLN or pol ν) is a newly discovered A family polymerase that generates a high error rate when incorporating nucleotides opposite dG; its translesion DNA synthesis (TLS) capability has only been demonstrated for high fidelity replication bypass of thymine glycol lesions. In the current investigation, we describe a novel TLS substrate specificity of pol ν, demonstrating that it is able to bypass exceptionally large DNA lesions whose linkages are through the DNA major groove. Specifically, pol ν catalyzed efficient and high fidelity TLS past peptides linked to N6-dA via a reduced Schiff base linkage with a γ-hydroxypropano-dA. Additionally, pol ν could bypass DNA interstrand cross-links with linkage between N6-dAs in complementary DNA strands. However, the chemically identical DNA−peptide and DNA interstrand cross-links completely blocked pol ν when they were located in the minor groove via a N2-dG linkage. Furthermore, we showed that pol ν incorporated a nucleotide opposite the 1,N6-etheno-dA (εdA) in an error-free manner and (+)-trans-anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide-dA [(+)-BPDE-dA] in an error-prone manner, albeit with a greatly reduced capability. Collectively, these data suggest that although pol ν bypass capacity cannot be generalized to all major groove DNA adducts, this polymerase could be involved in TLS when genomic replication is blocked by extremely large major groove DNA lesions. In view of the recent observation that pol ν may have a role in cellular tolerance to DNA cross-linking agents, our findings provide biochemical evidence for the potential functioning of this polymerase in the bypass of some DNA−protein and DNA−DNA cross-links.

Human DNA Polymerase ι Promotes Replication through a Ring-Closed Minor-Groove Adduct That Adopts a syn Conformation in DNA

ABSTRACT Acrolein, an α,β-unsaturated aldehyde, is generated in vivo as the end product of lipid peroxidation and from oxidation of polyamines. The reaction of acrolein with the N2 group of guanine in DNA leads to the formation of a cyclic adduct, γ-hydroxy-1,N 2-propano-2′-deoxyguanosine (γ-HOPdG). Previously, we have shown that proficient replication through the γ-HOPdG adduct can be mediated by the sequential action of human DNA polymerases (Pols) ι and κ, in which Polι incorporates either pyrimidine opposite γ-HOPdG, but Polκ extends only from the cytosine. Since γ-HOPdG can adopt either a ring-closed cyclic form or a ring-opened form in DNA, to better understand the mechanisms that Pols ι and κ employ to promote replication through this lesion, we have examined the ability of these polymerases to replicate through the structural analogs of γ-HOPdG that are permanently either ring closed or ring opened. Our studies with these model adducts show that whereas the ring-opened form of γ-HOPdG is not inhibitory to synthesis by human Pols η, ι, or κ, only Polι is able to incorporate nucleotides opposite the ring-closed form, which is known to adopt a syn conformation in DNA. From these studies, we infer that (i) Pols η, ι, and κ have the ability to proficiently replicate through minor-groove DNA lesions that do not perturb the Watson-Crick hydrogen bonding of the template base with the incoming nucleotide, and (ii) Polι can accommodate a minor-groove-adducted template purine which adopts a syn conformation in DNA and forms a Hoogsteen base pair with the incoming nucleotide.