Human abasic endonuclease action on multilesion abasic clusters: implications for radiation-induced biological damage

Clustered damages—two or more closely opposed abasic sites, oxidized bases or strand breaks—are induced in DNA by ionizing radiation and by some radiomimetic drugs. They are potentially mutagenic or lethal. High complexity, multilesion clusters (three or more lesions) are hypothesized as repair-resistant and responsible for the greater biological damage induced by high linear energy transfer radiation (e.g. charged particles) than by low linear energy transfer X- or γ-rays. We tested this hypothesis by assessing human abasic endonuclease Ape1 activity on two- and multiple-lesion abasic clusters. We constructed cluster-containing oligonucleotides using a central variable cassette with abasic site(s) at specific locations, and 5′ and 3′ terminal segments tagged with visually distinctive fluorophores. The results indicate that in two- or multiple-lesion clusters, the spatial arrangement of uni-sided positive [in which the opposing strand lesion(s) is 3′ to the base opposite the reference lesion)] or negative polarity [opposing strand lesion(s) 5′ to the base opposite the reference lesion] abasic clusters is key in determining Ape1 cleavage efficiency. However, no bipolar clusters (minimally three-lesions) were good Ape1 substrates. The data suggest an underlying molecular mechanism for the higher levels of biological damage associated with agents producing complex clusters: the induction of highly repair-resistant bipolar clusters.     

Structural insights into abasic site for Fpg specific binding and catalysis: comparative high-resolution crystallographic studies of Fpg bound to various models of abasic site analogues-containing DNA

Fpg is a DNA glycosylase that recognizes and excises the mutagenic 8-oxoguanine (8-oxoG) and the potentially lethal formamidopyrimidic residues (Fapy). Fpg is also associated with an AP lyase activity which successively cleaves the abasic (AP) site at the 3′ and 5′ sides by βδ-elimination. Here, we present the high-resolution crystal structures of the wild-type and the P1G defective mutant of Fpg from Lactococcus lactis bound to 14mer DNA duplexes containing either a tetrahydrofuran (THF) or 1,3-propanediol (Pr) AP site analogues. Structures show that THF is less extrahelical than Pr and its backbone C5′–C4′–C3′ diverges significantly from those of Pr, rAP, 8-oxodG and FapydG. Clearly, the heterocyclic oxygen of THF is pushed back by the carboxylate of the strictly conserved E2 residue. We can propose that the ring-opened form of the damaged deoxyribose is the structure active form of the sugar for Fpg catalysis process. Both structural and functional data suggest that the first step of catalysis mediated by Fpg involves the expulsion of the O4′ leaving group facilitated by general acid catalysis (involving E2), rather than the immediate cleavage of the N-glycosic bond of the damaged nucleoside.     

Solution structure of an oligonucleotide containing an abasic site: evidence for an unusual deoxyribose conformation

The antitumor antibiotic bleomycin causes two major lesions in the deoxyribose backbone of DNA: formation of 4′-keto abasic sites and formation of strand breaks with 3′-phosphoglycolate and 5′-phosphate ends. As a model for the 4′-keto abasic site, we have characterized an abasic site (X) in d(CCAAAGXACTGGG)·d(CCCAGTACTTTGG) by two-dimensional NMR spectroscopy. A total of 475 NOEs and 101 dihedral angles provided the restraints for molecular modeling. Four unusual NOEs were observed between each anomer of the abasic site and the neighboring bases. In addition, four coupling constants for adjacent protons of the deoxyribose of both the α and β anomers of the abasic site were observed. The modeling suggests that for both anomers the abasic site is extrahelical, without significant distortion of the backbone opposite the lesion. The coupling constants further allowed assignment of an unusual sugar pucker for each anomer. The unique position of the abasic site in our structural model for each anomer is discussed in terms of repair of such lesions in vivo.     

Quantitative analysis of the efficiency and mutagenic spectra of abasic lesion bypass catalyzed by human Y-family DNA polymerases

Higher eukaryotes encode various Y-family DNA polymerases to perform global DNA lesion bypass. To provide complete mutation spectra for abasic lesion bypass, we employed short oligonucleotide sequencing assays to determine the sequences of abasic lesion bypass products synthesized by human Y-family DNA polymerases eta (hPolη), iota (hPolι) and kappa (hPolκ). The fourth human Y-family DNA polymerase, Rev1, failed to generate full-length lesion bypass products after 3 h. The results indicate that hPolι generates mutations with a frequency from 10 to 80% during each nucleotide incorporation event. In contrast, hPolη is the least error prone, generating the fewest mutations in the vicinity of the abasic lesion and inserting dAMP with a frequency of 67% opposite the abasic site. While the error frequency of hPolκ is intermediate to those of hPolη and hPolι, hPolκ has the highest potential to create frameshift mutations opposite the abasic site. Moreover, the time (t₅₀^bypass) required to bypass 50% of the abasic lesions encountered by hPolη, hPolι and hPolκ was 4.6, 112 and 1 823 s, respectively. These t₅₀^bypass values indicate that, among the enzymes, hPolη has the highest abasic lesion bypass efficiency. Together, our data suggest that hPolη is best suited to perform abasic lesion bypass in vivo.     

High efficiency detection of bi-stranded abasic clusters in gamma-irradiated DNA by putrescine

Bi-stranded abasic clusters, an abasic (AP) site on one DNA strand and another nearby AP site or strand break on the other, have been quantified using Nfo protein from Escherichia coli to produce a double-strand break at cluster sites. Since recent data suggest that Nfo protein cleaves inefficiently at some clusters, we tested whether polyamines, which also cut at AP sites, would cleave abasic clusters at higher efficiency. The data show that Nfo protein cleaves poorly at clusters containing immediately opposed AP sites and those separated by 1 or 3 bp. Putrescine (PUTR) cleaved more efficiently than spermidine or spermine, and did not cleave undamaged DNA. It cleaved abasic clusters in oligonucleotide duplexes more effectively than Nfo protein, including immediately opposed or closely spaced clusters. PUTR cleaved more efficiently than Nfo protein by a factor of ~1.7 or ~2 for DNA that had been γ-irradiated in moderate or non-radioquenching conditions, respectively. This suggests that the DNA environment during irradiation affects the spectrum of cluster configurations. Further comparison of PUTR and Nfo protein cleavage may provide useful information on abasic cluster levels and configurations induced by ionizing radiation.     

Structural and Kinetic Analysis of Nucleoside Triphosphate Incorporation Opposite an Abasic Site by Human Translesion DNA Polymerase η

The most common lesion in DNA is an abasic site resulting from glycolytic cleavage of a base. In a number of cellular studies, abasic sites preferentially code for dATP insertion (the “A rule”). In some cases frameshifts are also common. X-ray structures with abasic sites in oligonucleotides have been reported for several microbial and human DNA polymerases (pols), e.g. Dpo4, RB69, KlenTaq, yeast pol ι, human (h) pol ι, and human pol β. We reported previously that hpol η is a major pol involved in abasic site bypass (Choi, J.-Y., Lim, S., Kim, E. J., Jo, A., and Guengerich, F. P. (2010 J. Mol. Biol. 404, 34–44). hpol η inserted all four dNTPs in steady-state and pre-steady-state assays, preferentially inserting A and G. In LC-MS analysis of primer-template pairs, A and G were inserted but little C or T was inserted. Frameshifts were observed when an appropriate pyrimidine was positioned 5′ to the abasic site in the template. In x-ray structures of hpol η with a non-hydrolyzable analog of dATP or dGTP opposite an abasic site, H-bonding was observed between the phosphate 5′ to the abasic site and water H-bonded to N1 and N6 of A and N1 and O6 of G nucleoside triphosphate analogs, offering an explanation for what appears to be a “purine rule.” A structure was also obtained for an A inserted and bonded in the primer opposite the abasic site, but it did not pair with a 5′ T in the template. We conclude that hpol η, a major copying enzyme with abasic sites, follows a purine rule, which can also lead to frameshifts. The phenomenon can be explained with H-bonds.     

Escherichia coli HU protein has a role in the repair of abasic sites in DNA

HU is one of the most abundant DNA binding proteins in Escherichia coli. We find that it binds strongly to DNA containing an abasic (AP) site or tetrahydrofuran (THF) (apparent K_d ≈50 nM). It also possesses an AP lyase activity that cleaves at a deoxyribose but not at a THF residue. The binding and cleavage of an AP site was observed only with the HUαβ heterodimer. Site-specific mutations at K3 and R61 residues led to a change in substrate binding and cleavage. Both K3A(α)K3A(β) and R61A(α)R61A(β) mutant HU showed significant reduction in binding to DNA containing AP site; however, only R61A(α)R61A(β) mutant protein exhibited significant loss in AP lyase activity. Both K3A(α)K3A(β) and R61K(α)R61K(β) showed slight reduction in AP lyase activities. The function of HU protein as an AP lyase was confirmed by the ability of hupA or hupB mutations to further reduce the viability of an E. coli dut(Ts) xth mutant, which generates lethal AP sites at 37°C; the hupA and hupB derivatives, respectively, had a 6-fold and a 150-fold lower survival at 37°C than did the parental strain. These data suggest, therefore, that HU protein plays a significant role in the repair of AP sites in E. coli.     

Blocked and unblocked 5' termini in reovirus genome RNA

Uniformly ³²P-labeled, double-stranded genome RNA isolated from purified reovirus contains two types of 5′-terminal sequences. One strand contains a phosphatase-resistant 5′-terminal structure, XpppG^*pCpU, which is also present in the viral mRNA. The 5′ blocking group, X, is removed by β-elimination indicating that it is a nucleoside containing free 2′,3′-hydroxyls. G^*pC is an alkaline-resistant, 2′-O-methylated sequence. The other strand contains a phosphatase-sensitive 5′ sequence, ppGpPupPyp. The results are discussed in relation to blocked 5′-terminal structures in other viral and cellular RNAs.     

Sensitivity of human type II topoisomerases to DNA damage: stimulation of enzyme-mediated DNA cleavage by abasic, oxidized and alkylated lesions

Type II topoisomerases are essential enzymes that are also the primary cellular targets for a number of important anticancer drugs. These drugs act by increasing levels of topoisomerase II-mediated DNA cleavage. Recent studies indicate that endogenous forms of DNA damage, such as abasic sites and base mismatches, also stimulate the DNA scission activity of the enzyme. To extend our understanding of how type II topoisomerases react to DNA damage, the effects of abasic sites, and oxidized and alkylated bases on DNA cleavage mediated by human topo-isomerase IIα and β were determined. Based on experiments that incorporated random abasic sites into plasmid DNA, human type II enzymes can locate lesions even within a background of several thousand undamaged base pairs. As determined by experiments that utilized site-specific forms of DNA lesions, oxidized or monoalkylated purines that allow base pairing and induce little distortion in the double helix have modest effects on topoisomerase II-mediated DNA cleavage. In contrast, 1,N⁶-ethenoadenine, a bulky lesion that disrupts base pairing, enhanced DNA cleavage ~10-fold. 1,N⁶-Ethenoadenine is the first lesion found to rival the stimulatory effects of apurinic sites on the DNA scission activity of eukaryotic type II topoisomerases.     

Mutagenic effects of abasic and oxidized abasic lesions in Saccharomyces cerevisiae

2-Deoxyribonolactone (L) and 2-deoxyribose (AP) are abasic sites that are produced by ionizing radiation, reactive oxygen species and a variety of DNA damaging agents. The biological processing of the AP site has been examined in the yeast Saccharomyces cerevisiae. However, nothing is known about how L is processed in this organism. We determined the bypass and mutagenic specificity of DNA containing an abasic site (AP and L) or the AP analog tetrahydrofuran (F) using an oligonucleotide transformation assay. The tetrahydrofuran analog and L were bypassed at 10-fold higher frequencies than the AP lesions. Bypass frequencies of lesions were greatly reduced in the absence of Rev1 or Polζ (rev3 mutant), but were only marginally reduced in the absence of Polη (rad30 mutant). Deoxycytidine was the preferred nucleotide inserted opposite an AP site whereas dA and dC were inserted at equal frequencies opposite F and L sites. In the rev1 and rev3 strains, dA was the predominant nucleotide inserted opposite these lesions. Overall, we conclude that both Rev1 and Polζ are required for the efficient bypass of abasic sites in yeast.     

Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2′-fluoro-ANA/RNA and DNA/RNA hybrids

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2′-fluoro-ANA analog (2′F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2′F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3′-endo (north, A-form) conformation, whereas those of the ANA strand adopt a ‘rigid’ O4′-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2′F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2′F-ANA/RNA and DNA/RNA helices is 9.0 ± 0.5 Å, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2′F-ANA/RNA hybrids to elicit RNase H activity.     

Coordinated Processing of 3′ Slipped (CAG)n/(CTG)n Hairpins by DNA Polymerases β and δ Preferentially Induces Repeat Expansions

Expansion of CAG/CTG trinucleotide repeats causes certain familial neurological disorders. Hairpin formation in the nascent strand during DNA synthesis is considered a major path for CAG/CTG repeat expansion. However, the underlying mechanism is unclear. We show here that removal or retention of a nascent strand hairpin during DNA synthesis depends on hairpin structures and types of DNA polymerases. Polymerase (pol) δ alone removes the 3′-slipped hairpin using its 3′-5′ proofreading activity when the hairpin contains no immediate 3′ complementary sequences. However, in the presence of pol β, pol δ preferentially facilitates hairpin retention regardless of hairpin structures. In this reaction, pol β incorporates several nucleotides to the hairpin 3′-end, which serves as an effective primer for the continuous DNA synthesis by pol δ, thereby leading to hairpin retention and repeat expansion. These findings strongly suggest that coordinated processing of 3′-slipped (CAG)_n/(CTG)_n hairpins by polymerases δ and β on during DNA synthesis induces CAG/CTG repeat expansions.     

Processing of bistranded abasic DNA clusters in gamma-irradiated human hematopoietic cells

Clustered DNA damages—two or more lesions on opposing strands and within one or two helical turns—are formed in cells by ionizing radiation or radiomimetic antitumor drugs. They are hypothesized to be difficult to repair, and thus are critical biological damages. Since individual abasic sites can be cytotoxic or mutagenic, abasic DNA clusters are likely to have significant cellular impact. Using a novel approach for distinguishing abasic clusters that are very closely spaced (putrescine cleavage) or less closely spaced (Nfo protein cleavage), we measured induction and processing of abasic clusters in 28SC human monocytes that were exposed to ionizing radiation. γ-rays induced ~1 double-strand break: 1.3 putrescine-detected abasic clusters: 0.8 Nfo-detected abasic clusters. After irradiation, the 28SC cells rejoined double-strand breaks efficiently within 24 h. In contrast, in these cells, the levels of abasic clusters decreased very slowly over 14 days to background levels. In vitro repair experiments that used 28SC cell extracts further support the idea of slow processing of specific, closely spaced abasic clusters. Although some clusters were removed by active cellular repair, a substantial number was apparently decreased by ‘splitting’ during DNA replication and subsequent cell division. The existence of abasic clusters in 28SC monocytes, several days after irradiation suggests that they constitute persistent damages that could lead to mutation or cell killing.     

Human TDP1, APE1 and TREX1 repair 3′-DNA–peptide/protein cross-links arising from abasic sites in vitro

Histones and many other proteins react with abundant endogenous DNA lesions, apurinic/apyrimidinic (abasic, AP) sites and/or 3′-phospho-α,β-unsaturated aldehyde (3′-PUA), to form unstable but long-lived Schiff base DNA–protein cross-links at 3′-DNA termini (3′-PUA–protein DPCs). Poly (ADP-ribose) polymerase 1 (PARP1) cross-links to the AP site in a similar manner but the Schiff base is reduced by PARP1’s intrinsic redox capacity, yielding a stable 3′-PUA–PARP1 DPC. Eradicating these DPCs is critical for maintaining the genome integrity because 3′-hydroxyl is required for DNA synthesis and ligation. But how they are repaired is not well understood. Herein, we chemically synthesized 3′-PUA-aminooxylysine-peptide adducts that closely resemble the proteolytic 3′-PUA–protein DPCs, and found that they can be repaired by human tyrosyl-DNA phosphodiesterase 1 (TDP1), AP endonuclease 1 (APE1) and three-prime repair exonuclease 1 (TREX1). We characterized these novel repair pathways by measuring the kinetic constants and determining the effect of cross-linked peptide length, flanking DNA structure, and the opposite nucleobase. We further found that these nucleases can directly repair 3′-PUA–histone DPCs, but not 3′-PUA–PARP1 DPCs unless proteolysis occurs initially. Collectively, we demonstrated that in vitro 3′-PUA–protein DPCs can be repaired by TDP1, APE1, and TREX1 following proteolysis, but the proteolysis is not absolutely required for smaller DPCs.     

Strand displacement synthesis by yeast DNA polymerase ε

DNA polymerase ε (Pol ε) is a replicative DNA polymerase with an associated 3′–5′ exonuclease activity. Here, we explored the capacity of Pol ε to perform strand displacement synthesis, a process that influences many DNA transactions in vivo. We found that Pol ε is unable to carry out extended strand displacement synthesis unless its 3′–5′ exonuclease activity is removed. However, the wild-type Pol ε holoenzyme efficiently displaced one nucleotide when encountering double-stranded DNA after filling a gap or nicked DNA. A flap, mimicking a D-loop or a hairpin structure, on the 5′ end of the blocking primer inhibited Pol ε from synthesizing DNA up to the fork junction. This inhibition was observed for Pol ε but not with Pol δ, RB69 gp43 or Pol η. Neither was Pol ε able to extend a D-loop in reconstitution experiments. Finally, we show that the observed strand displacement synthesis by exonuclease-deficient Pol ε is distributive. Our results suggest that Pol ε is unable to extend the invading strand in D-loops during homologous recombination or to add more than two nucleotides during long-patch base excision repair. Our results support the hypothesis that Pol ε participates in short-patch base excision repair and ribonucleotide excision repair.     

A 5′, 8-cyclo-2′-deoxypurine lesion induces trinucleotide repeat deletion via a unique lesion bypass by DNA polymerase β

5′,8-cyclo-2′-deoxypurines (cdPus) are common forms of oxidized DNA lesions resulting from endogenous and environmental oxidative stress such as ionizing radiation. The lesions can only be repaired by nucleotide excision repair with a low efficiency. This results in their accumulation in the genome that leads to stalling of the replication DNA polymerases and poor lesion bypass by translesion DNA polymerases. Trinucleotide repeats (TNRs) consist of tandem repeats of Gs and As and therefore are hotspots of cdPus. In this study, we provided the first evidence that both (5′R)- and (5′S)-5′,8-cyclo-2′-deoxyadenosine (cdA) in a CAG repeat tract caused CTG repeat deletion exclusively during DNA lagging strand maturation and base excision repair. We found that a cdA induced the formation of a CAG loop in the template strand, which was skipped over by DNA polymerase β (pol β) lesion bypass synthesis. This subsequently resulted in the formation of a long flap that was efficiently cleaved by flap endonuclease 1, thereby leading to repeat deletion. Our study indicates that accumulation of cdPus in the human genome can lead to TNR instability via a unique lesion bypass by pol β.     

DNA oligonucleotides with A, T, G or C opposite an abasic site: structure and dynamics

Abasic sites are common DNA lesions resulting from spontaneous depurination and excision of damaged nucleobases by DNA repair enzymes. However, the influence of the local sequence context on the structure of the abasic site and ultimately, its recognition and repair, remains elusive. In the present study, duplex DNAs with three different bases (G, C or T) opposite an abasic site have been synthesized in the same sequence context (5′-CCA AAG₆ XA₈C CGG G-3′, where X denotes the abasic site) and characterized by 2D NMR spectroscopy. Studies on a duplex DNA with an A opposite the abasic site in the same sequence has recently been reported [Chen,J., Dupradeau,F.-Y., Case,D.A., Turner,C.J. and Stubbe,J. (2007) Nuclear magnetic resonance structural studies and molecular modeling of duplex DNA containing normal and 4′-oxidized abasic sites. Biochemistry, 46, 3096–3107]. Molecular modeling based on NMR-derived distance and dihedral angle restraints and molecular dynamics calculations have been applied to determine structural models and conformational flexibility of each duplex. The results indicate that all four duplexes adopt an overall B-form conformation with each unpaired base stacked between adjacent bases intrahelically. The conformation around the abasic site is more perturbed when the base opposite to the lesion is a pyrimidine (C or T) than a purine (G or A). In both the former cases, the neighboring base pairs (G6-C21 and A8-T19) are closer to each other than those in B-form DNA. Molecular dynamics simulations reveal that transient H-bond interactions between the unpaired pyrimidine (C20 or T20) and the base 3′ to the abasic site play an important role in perturbing the local conformation. These results provide structural insight into the dynamics of abasic sites that are intrinsically modulated by the bases opposite the abasic site.     

Lack of sugar discrimination by human Pol mu requires a single glycine residue

DNA polymerase mu (Pol µ) is a novel family X DNA polymerase that has been suggested to play a role in micro-homology mediated joining and repair of double strand breaks. We show here that human Pol µ is not able to discriminate against the 2′-OH group of the sugar moiety. It inserts rNTPs with an efficiency that is <10-fold lower than that of dNTPs, in sharp contrast with the >1000-fold discrimination characteristic of most DNA-dependent DNA polymerases. The lack of sugar discrimination by Pol µ is demonstrated by its ability to add rNTPs to both DNA and RNA primer strands, and to insert both deoxy- and ribonucleotides on growing nucleic acid chains. 3D-modelling of human Pol µ based on the available Pol β and TdT structural information allowed us to predict candidate residues involved in sugar discrimination. Thus, a single amino acid substitution in which Gly433 residue of Pol µ was mutated to the consensus tyrosine present in Pol β, produced a strong increase in the discrimination against ribonucleotides. The unusual capacity to insert both rNTPs and dNTPs will be discussed in the context of the predicted roles of Pol µ in DNA repair.