R. Stephen Lloyd

MutY catalytic core, mutant and bound adenine structures define specificity for DNA repair enzyme superfamily.

The DNA glycosylase MutY, which is a member of the Helix-hairpin-Helix (HhH) DNA glycosylase superfamily, excises adenine from mispairs with 8-oxoguanine and guanine. High-resolution crystal structures of the MutY catalytic core (cMutY), the complex with bound adenine, and designed mutants reveal the basis for adenine specificity and glycosyl bond cleavage chemistry. The two cMutY helical domains form a positively-charged groove with the adenine-specific pocket at their interface. The Watson-Crick hydrogen bond partners of the bound adenine are substituted by protein atoms, confirming a nucleotide flipping mechanism, and supporting a specific DNA binding orientation by MutY and structurally related DNA glycosylases.

Evidence for an Imino Intermediate in the DNA Polymerase β Deoxyribose Phosphate Excision Reaction

A recent study demonstrated that rat DNA polymerase β (β-pol) releases 5′-deoxyribose phosphate (dRP) termini from preincised apurinic/apyrimidinic DNA, a substrate generated during certain types of base excision repair. This catalytic activity resides within the amino-terminal, 8-kDa domain of β-pol and occurs via β-elimination as opposed to hydrolysis (Matsumoto, Y., and Kim, K. (1995) Science 269, 699-702). The latter finding suggested that the dRP excision reaction might proceed via an imine intermediate. In order to test this hypothesis, we attempted to trap β-pol on preincised apurinic/apyrimidinic DNA using NaBH4 as the reducing agent. Both 8-kDa domain-DNA and intact β-pol-DNA complexes were detected and identified by autoradiography coupled to immunoblotting. Our results indicate that the chemical mechanism of the β-pol dRpase reaction does proceed through an imine enzyme-DNA intermediate and that the active site residue responsible for dRP release must therefore contain a primary amine.

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.

Targeted deletion of the genes encoding NTH1 and NEIL1 DNA N-glycosylases reveals the existence of novel carcinogenic oxidative damage to DNA ☆

We have generated a strain of mice lacking two DNA N-glycosylases of base excision repair (BER), NTH1 and NEIL1, homologs of bacterial Nth (endonuclease three) and Nei (endonuclease eight). Although these enzymes remove several oxidized bases from DNA, they do not remove the well-known carcinogenic oxidation product of guanine: 7,8-dihydro-8-oxoguanine (8-OH-Gua), which is removed by another DNA N-glycosylase, OGG1. The Nth1-/-Neil1-/- mice developed pulmonary and hepatocellular tumors in much higher incidence than either of the single knockouts, Nth1-/- and Neil1-/-. The pulmonary tumors contained, exclusively, activating GGT-->GAT transitions in codon 12 of K-ras of their DNA. Such transitions contrast sharply with the activating GGT-->GTT transversions in codon 12 of K-ras of the pathologically similar pulmonary tumors, which arose in mice lacking OGG1 and a second DNA N-glycosylase, MUTY. To characterize the biochemical phenotype of the knockout mice, the content of oxidative DNA base damage was analyzed from three tissues isolated from control, single and double knockout mice. The content of 8-OH-Gua was indistinguishable among all genotypes. In contrast, the content of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) derived from adenine and guanine, respectively, were increased in some but not all tissues of Neil1-/- and Neil1-/-Nth1-/- mice. The high incidence of tumors in our Nth1-/-Neil1-/- mice together with the nature of the activating mutation in the K-ras gene of their pulmonary tumors, reveal for the first time, the existence of mutagenic and carcinogenic oxidative damage to DNA which is not 8-OH-Gua.

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.

Identification of the Structural and Functional Domains of MutY, an Escherichia coli DNA Mismatch Repair Enzyme

The linear amino acid sequences of the Escherichia coli DNA repair proteins, MutY and endonuclease III, show significant homology, even though these enzymes recognize entirely different substrates. In this study, proteolysis and molecular modeling of MutY were used to elucidate its domain organization. Proteolysis by trypsin cleaved the enzyme into 26- and 13-kDa fragments. NH2-terminal sequencing showed that the p13 domain begins at Gln226, indicating that the COOH-terminal portion of MutY, absent in endonuclease III, is organized as a separate domain. The large p26 domain is almost equivalent to the size of endonuclease III. Binding activity of the p26 domain to a DNA substrate containing an A·G mismatch was comparable with that of the intact enzyme. In vitro studies show that the p26 domain retains adenine glycosylase and AP lyase activity on DNA containing undamaged adenine opposite guanine or 8-oxo-7,8-dihydro-2′-deoxyguanine. Although the activity was somewhat reduced, the above results show that the critical amino acid residues involved in substrate binding and catalysis are present in this domain. The structure predicted by molecular modeling indicates that the region of MutY (Met1-Trp216), which is homologous to endonuclease III exhibits a two domain structure, even though this portion is resistant to proteolysis by trypsin.

Processivity of uracil DNA glycosylase

The purpose of this study was to determine the mechanism by which uracil DNA glycosylase locates uracil residues within double-stranded DNA. Using reaction conditions that contained low salt concentrations, the addition of uracil DNA glycosylase to plasmid DNAs containing multiple, randomly incorporated uracils resulted in the accumulation of form III DNA while unreacted form I DNA was still present. These data suggested that the enzyme utilizes a one-dimensional DNA-scanning mechanism such that this linear DNA arose by the accumulation of many single-strand breaks within the plasmid prior to enzyme dissociation. Reactions containing higher concentrations of uracil DNA glycosylase revealed a further accumulation of form III DNA after all form I DNA had been lost. These results suggested a partial (1.5-2 kb) enzyme processivity since the enzyme does not incise at all uracil bases on the DNA molecule prior to dissociation from that DNA. Since DNA scanning is regulated by electrostatic interactions, the processivity of the enzyme was evaluated through kinetic analyses of incision at various salt concentrations. At NaCl concentrations (< 50 mM), a significant amount of form III DNA accumulated while there were still unreacted form I DNAs present. In contrast, the accumulation of form III DNA was delayed at higher salt concentrations and the overall accumulation of form III DNA was less than that monitored at lower salt concentrations. DNAs were also analyzed by denaturing agarose gel electrophoresis in order to measure the average distance between strand breaks. Southern hybridizations showed a greater accumulation of breaks in DNAs that were reacted with the uracil DNA glycosylase at the lower salt concentrations, confirming a partial processivity for the enzyme.