HMCES Maintains Genome Integrity by Shielding Abasic Sites in Single-Strand DNA.

Abasic sites are one of the most common DNA lesions. All known abasic site repair mechanisms operate only when the damage is in double-stranded DNA. Here, we report the discovery of 5-hydroxymethylcytosine (5hmC) binding, ESC-specific (HMCES) as a sensor of abasic sites in single-stranded DNA. HMCES acts at replication forks, binds PCNA and single-stranded DNA, and generates a DNA-protein crosslink to shield abasic sites from error-prone processing. This unusual HMCES DNA-protein crosslink intermediate is resolved by proteasome-mediated degradation. Acting as a suicide enzyme, HMCES prevents translesion DNA synthesis and the action of endonucleases that would otherwise generate mutations and double-strand breaks. HMCES is evolutionarily conserved in all domains of life, and its biochemical properties are shared with its E. coli ortholog. Thus, HMCES is an ancient DNA lesion recognition protein that preserves genome integrity by promoting error-free repair of abasic sites in single-stranded DNA.

Genomic landscape of oxidative DNA damage and repair reveals regioselective protection from mutagenesis.

BACKGROUND: DNA is subject to constant chemical modification and damage, which eventually results in variable mutation rates throughout the genome. Although detailed molecular mechanisms of DNA damage and repair are well understood, damage impact and execution of repair across a genome remain poorly defined. RESULTS: To bridge the gap between our understanding of DNA repair and mutation distributions, we developed a novel method, AP-seq, capable of mapping apurinic sites and 8-oxo-7,8-dihydroguanine bases at approximately 250-bp resolution on a genome-wide scale. We directly demonstrate that the accumulation rate of apurinic sites varies widely across the genome, with hot spots acquiring many times more damage than cold spots. Unlike single nucleotide variants (SNVs) in cancers, damage burden correlates with marks for open chromatin notably H3K9ac and H3K4me2. Apurinic sites and oxidative damage are also highly enriched in transposable elements and other repetitive sequences. In contrast, we observe a reduction at chromatin loop anchors with increased damage load towards inactive compartments. Less damage is found at promoters, exons, and termination sites, but not introns, in a seemingly transcription-independent but GC content-dependent manner. Leveraging cancer genomic data, we also find locally reduced SNV rates in promoters, coding sequence, and other functional elements. CONCLUSIONS: Our study reveals that oxidative DNA damage accumulation and repair differ strongly across the genome, but culminate in a previously unappreciated mechanism that safeguards the regulatory and coding regions of genes from mutations.

Mcl-1 suppresses abasic site repair following bile acid-induced hepatic cellular DNA damage.

In cholestasis, increases in bile acid levels result in the generation of reactive oxygen species and the induction of DNA damage and mutation. It is believed that bile acid accumulation is associated with liver tumorigenesis. However, the mechanism that underpins this phenomenon remains to be elucidated. Mcl-1, which is overexpressed in hepatic cells, is a pro-survival member of the Bcl-2 family. In this study, we observed that Mcl-1 potently suppresses the repair of bile acid-induced abasic (apurinic/apyrimidinic) sites in DNA lesions. Upon exposure of hepatic cells to glycochenodeoxycholate, one of the major conjugated human bile acids, we observed an increase in AP site accumulation along with induction of poly(ADP-ribose) polymerase and XRCC1 ( X-Ray Repair Cross Complementing 1). In addition, accumulation of Mcl-1 was observed in the nuclei of QGY-7703 cells in response to glycochenodeoxycholate stimulation. Knockdown of endogenous Mcl-1 by RNA interference significantly accelerated the repair of DNA lesions in glycochenodeoxycholate-treated cells. However, unlike XRCC1, poly(ADP-ribose) polymerase was induced following Mcl-1 knockdown. Conversely, poly(ADP-ribose) polymerase suppression was observed following glycochenodeoxycholate treatment of cells overexpressing Mcl-1. Moreover, AP-site counting analyses revealed that DNA repair activity was enhanced in cells overexpressing poly(ADP-ribose) polymerase under glycochenodeoxycholate stress conditions. It is well known that poly(ADP-ribose) polymerase plays a crucial role in the base excision repair pathway. Thus, our findings suggest that Mcl-1 suppresses base excision repair by inhibiting poly(ADP-ribose) polymerase induction following glycochenodeoxycholate-induced DNA damage. These results potentially explain how bile acid accumulation results in genetic instability and carcinogenesis.

Interstrand cross-links arising from strand breaks at true abasic sites in duplex DNA.

Interstrand cross-links are exceptionally bioactive DNA lesions. Endogenous generation of interstrand cross-links in genomic DNA may contribute to aging, neurodegeneration, and cancer. Abasic (Ap) sites are common lesions in genomic DNA that readily undergo spontaneous and amine-catalyzed strand cleavage reactions that generate a 2,3-didehydro-2,3-dideoxyribose sugar remnant (3'ddR5p) at the 3'-terminus of the strand break. Interestingly, this strand scission process leaves an electrophilic α,ß-unsaturated aldehyde residue embedded within the resulting nicked duplex. Here we present evidence that 3'ddR5p derivatives generated by spermine-catalyzed strand cleavage at Ap sites in duplex DNA can react with adenine residues on the opposing strand to generate a complex lesion consisting of an interstrand cross-link adjacent to a strand break. The cross-link blocks DNA replication by ϕ29 DNA polymerase, a highly processive polymerase enzyme that couples synthesis with strand displacement. This suggests that 3'ddR5p-derived cross-links have the potential to block critical cellular DNA transactions that require strand separation. LC-MS/MS methods developed herein provide powerful tools for studying the occurrence and properties of these cross-links in biochemical and biological systems.

Replication and repair of a reduced 2΄-deoxyguanosine-abasic site interstrand cross-link in human cells.

Apurinic/apyrimidinic (AP) sites, or abasic sites, which are a common type of endogenous DNA damage, can forge interstrand DNA-DNA cross-links via reaction with the exocyclic amino group on a nearby 2΄-deoxyguanosine or 2΄-deoxyadenosine in the opposite strand. Here, we utilized a shuttle vector method to examine the efficiency and fidelity with which a reduced dG-AP cross-link-containing plasmid was replicated in cultured human cells. Our results showed that the cross-link constituted strong impediments to DNA replication in HEK293T cells, with the bypass efficiencies for the dG- and AP-containing strands being 40% and 20%, respectively. While depletion of polymerase (Pol) η did not perturb the bypass efficiency of the lesion, the bypass efficiency was markedly reduced (to 1-10%) in the isogenic cells deficient in Pol κ, Pol ι or Pol ζ, suggesting the mutual involvement of multiple translesion synthesis polymerases in bypassing the lesion. Additionally, replication of the cross-linked AP residue in HEK293T cells was moderately error-prone, inducing a total of ∼26% single-nucleobase substitutions at the lesion site, whereas replication past the cross-linked dG component occurred at a mutation frequency of ∼8%. Together, our results provided important insights into the effects of an AP-derived interstrand cross-link on the efficiency and accuracy of DNA replication in human cells.

Pre-steady state kinetics of DNA binding and abasic site hydrolysis by tyrosyl-DNA phosphodiesterase 1.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) processes DNA 3'-end-blocking modifications, possesses DNA and RNA 3'-nucleosidase activity and is also able to hydrolyze an internal apurinic/apyrimidinic (AP) site and its synthetic analogs. The mechanism of Tdp1 interaction with DNA was analyzed using pre-steady state stopped-flow kinetics with tryptophan, 2-aminopurine and Förster resonance energy transfer fluorescence detection. Phosphorothioate or tetramethyl phosphoryl guanidine groups at the 3'-end of DNA have been used to prevent 3'-nucleosidase digestion by Tdp1. DNA binding and catalytic properties of Tdp1 and its mutants H493R (Tdp1 mutant SCAN1) and H263A have been compared. The data indicate that the initial step of Tdp1 interaction with DNA includes binding of Tdp1 to the DNA ends followed by the 3'-nucleosidase reaction. In the case of DNA containing AP site, three steps of fluorescence variation were detected that characterize (i) initial binding the enzyme to the termini of DNA, (ii) the conformational transitions of Tdp1 and (iii) search for and recognition of the AP-site in DNA, which leads to the formation of the catalytically active complex and to the AP-site cleavage reaction. Analysis of Tdp1 interaction with single- and double-stranded DNA substrates shows that the rates of the 3'-nucleosidase and AP-site cleavage reactions have similar values in the case of single-stranded DNA, whereas in double-stranded DNA, the cleavage of the AP-site proceeds two times faster than 3'-nucleosidase digestion. Therefore, the data show that the AP-site cleavage reaction is an essential function of Tdp1 which may comprise an independent of AP endonuclease 1 AP-site repair pathway.

Probing Enhanced Double-Strand Break Formation at Abasic Sites within Clustered Lesions in Nucleosome Core Particles.

DNA is rapidly cleaved under mild alkaline conditions at apyrimidinic/apurinic sites, but the half-life is several weeks in phosphate buffer (pH 7.5). However, abasic sites are ∼100-fold more reactive within nucleosome core particles (NCPs). Histone proteins catalyze the strand scission, and at superhelical location 1.5, the histone H4 tail is largely responsible for the accelerated cleavage. The rate constant for strand scission at an abasic site is enhanced further in a nucleosome core particle when it is part of a bistranded lesion containing a proximal strand break. Cleavage of this form results in a highly deleterious double-strand break. This acceleration is dependent upon the position of the abasic lesion in the NCP and its structure. The enhancement in cleavage rate at an apurinic/apyrimidinic site rapidly drops off as the distance between the strand break and abasic site increases and is negligible once the two forms of damage are separated by 7 bp. However, the enhancement of the rate of double-strand break formation increases when the size of the gap is increased from one to two nucleotides. In contrast, the cleavage rate enhancement at 2-deoxyribonolactone within bistranded lesions is more modest, and it is similar in free DNA and nucleosome core particles. We postulate that the enhanced rate of double-strand break formation at bistranded lesions containing apurinic/apyrimidinic sites within nucleosome core particles is a general phenomenon and is due to increased DNA flexibility.

Catalysts of DNA Strand Cleavage at Apurinic/Apyrimidinic Sites.

Apurinic/apyrimidinic (AP) sites are constantly formed in cellular DNA due to instability of the glycosidic bond, particularly at purines and various oxidized, alkylated, or otherwise damaged nucleobases. AP sites are also generated by DNA glycosylases that initiate DNA base excision repair. These lesions represent a significant block to DNA replication and are extremely mutagenic. Some DNA glycosylases possess AP lyase activities that nick the DNA strand at the deoxyribose moiety via a ß- or ß,δ-elimination reaction. Various amines can incise AP sites via a similar mechanism, but this non-enzymatic cleavage typically requires high reagent concentrations. Herein, we describe a new class of small molecules that function at low micromolar concentrations as both ß- and ß,δ-elimination catalysts at AP sites. Structure-activity relationships have established several characteristics that appear to be necessary for the formation of an iminium ion intermediate that self-catalyzes the elimination at the deoxyribose ring.

Design of a New Fluorescent Oligonucleotide-Based Assay for a Highly Specific Real-Time Detection of Apurinic/Apyrimidinic Site Cleavage by Tyrosyl-DNA Phosphodiesterase 1.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) promotes catalytic scission of a phosphodiester bond between the 3'-end of DNA and the hydroxyl group of a tyrosine residue, as well as cleaving off a variety of other 3'-terminal phosphate-linked DNA substituents. We have shown recently that Tdp1 can initiate an apurinic/apyrimidinic (AP) site repair pathway that is independent from the one mediated by AP endonuclease 1 (APE1). Until recently, there was no method available of tracking the AP-site cleaving activity of Tdp1 by real-time fluorescence assay. In the present study we demonstrate a highly specific real-time detection of the AP-site cleaving activity of Tdp1 which allows one to distinguish it from the activity of APE1 by using a short hairpin oligonucleotide with a 1,12-dodecanediol loop, a 5'-fluorophore, and a 3'-quencher. Specific phosphodiesterase activity of Tdp1, which is usually able to remove quencher from the 3'-end of DNA, was suppressed in our approach by introducing a noncleavable phosphate group mimic between the 3'-end and the quencher. As a nondigestible 3'-phosphate analogue, we have used a new uncharged tetramethyl phosphoryl guanidine (Tmg) group, which is resistant to 3'-phosphodiesterase cleavage.

[The role of Ku antigen in the repair of apurinic/apyrimidinic sites in DNA].

Apurinic/apyrimidinic (AP) sites are some of the most frequent lesions in genomic DNA. It is widely accepted that, irrespective of their origin, AP sites are further processed by the base excision repair (BER) machinery, being the central intermediate of this process. Under special conditions, proteins, which recognize AP sites, are able to form covalent adducts with DNA. By combination of the cross-linking technique with mass-spectrometry analysis, Ku antigen (Ku)--the central player in nonhomologous end joining (NHEJ), the pathway of double-strand break (DSB) repair--was identified as a protein reactive to AP sites. Moreover, Ku was shown to be a 5'-dRP/AP lyase that acts near DSBs in NHEJ. The recent studies have demonstrated involvement of Ku in the different stages of BER. Here, Ku roles in NHEJ and BER pathways of DNA repair are overviewed.

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 ß. 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.

[Tyrosyl-DNA Phosphodiesterase 1 Is a New Player in Repair of Apurinic/Apyrimidinic Sites].

Genomic DNA is constantly damaged by the action of exogenous factors and endogenous reactive metabolites. Apurinic/apyrimidinic sites (AP sites), which occur as a result of DNA glycosylase induced or spontaneous hydrolysis of the N-glycosidic bonds, are the most common damages of DNA. The chemical reactivity of AP sites is the cause of DNA breaks, and DNA-protein and DNA-DNA crosslinks. Repair of AP sites is one of the most important mechanisms for maintaining genome stability. Despite the fact that the main participants of the AP site repair are very well studied, the new proteins that could be involved potentially in this process as "back up" players or perform certain specialized functions are being found. This review is dedicated to one of these proteins, tyrosyl-DNA phosphodiesterase 1 (Tdp1), for which we have recently shown that in addition to its main activity of specific cleavage of the tyrosyl-DNA bond formed via a covalent attachment of topoisomerase 1 (Top1) to DNA, Tdp1 is able to initiate the cleavage of the internal AP sites in DNA and their following repair. Tdp1 was discovered in Saccharomyces cerevisiae yeast as an enzyme hydrolyzing the covalent bond between tyrosyl residue of topoisomerase 1 and 3'-phosphate group in DNA. Tdp1 is the major enzyme which carries out the repair of the irreversible complexes of DNA and topoisomerase 1, which appear. in the presence of Top 1 inhibitors, such as camptothecin, therefore Tdp1 is a very important target for the development of inhibitors--anticancer drugs. Besides, Tdp1 hydrolyzes a wide range of 3'-terminal DNA modifications and the 3'-end nucleosides and its derivatives to form a 3'-phosphate. Tdp1 ability to cleave AP sites suggests its involvement in the base excision repair as an alternative enzyme to cleave AP sites instead of AP endonuclease 1--the major enzyme hydrolyzing AP sites in DNA repair process.

Detecting DNA depurination with solid-state nanopores.

Among the different types of DNA damage that occur endogenously in the cell, depurination is especially prevalent. These lesions can initiate mutagenesis and have been implicated in a variety of diseases, including cancer. Here, we demonstrate a new approach for the detection of depurination at the single-molecule scale using solid-state nanopores. We induce depurination in short duplex DNA using acidic conditions and observe that the presence of apurinic sites results in significantly slower dynamics during electrokinetic translocation. This procedure may be valuable as a diagnostic for in situ quantification of DNA depurination.

Analysis of DNA strand cleavage at abasic sites.

Abasic sites in DNA arise under a variety of circumstances, including destabilization of bases through oxidative stress, as an intermediate in base excision repair, and through spontaneous loss. Their persistence can yield a blockade to RNA transcription and DNA synthesis and can be a source of mutations. Organisms have developed an enzymatic means of repairing abasic sites in DNA that generally involves a DNA repair pathway that is initiated by a repair protein creating a phosphodiester break ("nick") adjacent to the site of base loss. Here we describe a method for analyzing the manner in which repair endonucleases differ in the way they create nicks in DNA and how to distinguish between them using cellular crude extracts.

Interaction of apurinic/apyrimidinic endonuclease 2 (Apn2) with Myh1 DNA glycosylase in fission yeast.

Oxidative DNA damage is repaired primarily by the base excision repair (BER) pathway in a process initiated by removal of base lesions or mismatched bases by DNA glycosylases. MutY homolog (MYH, MUTYH, or Myh1) is a DNA glycosylase which excises adenine paired with the oxidative lesion 8-oxo-7,8-dihydroguanine (8-oxoG, or G°), thus reducing G:C to T:A mutations. The resulting apurinic/apyrimidinic (AP) site is processed by an AP-endonuclease or a bifunctional glycosylase/lyase. We show here that the major Schizosaccharomyces pombe AP endonuclease, Apn2, binds to the inter-domain connector located between the N- and C-terminal domains of Myh1. This Myh1 inter-domain connector also interacts with the Hus1 subunit of the Rad9-Rad1-Hus1 checkpoint clamp. Mutagenesis studies indicate that Apn2 and Hus1 bind overlapping but different sequence motifs on Myh1. Mutation on I(261) of Myh1 reduces its interaction with Hus1, but only slightly attenuates its interaction with Apn2. However, E(262) of Myh1 is a key determinant for both Apn2 and Hus1 interactions. Like human APE1, Apn2 has 3'-phosphodiesterase activity. However, unlike hAPE1, Apn2 has a weak AP endonuclease activity which cleaves the AP sites generated by Myh1 glycosylase. Functionally, Apn2 stimulates Myh1 glycosylase activity and Apn2 phosphodiesterase activity is stimulated by Myh1. The cross stimulation of Myh1 and Apn2 enzymatic activities is dependent on their physical interaction. Thus, Myh1 and Apn2 constitute an initial BER complex.

Deletion of individual Ku subunits in mice causes an NHEJ-independent phenotype potentially by altering apurinic/apyrimidinic site repair.

Ku70 and Ku80 form a heterodimer called Ku that forms a holoenzyme with DNA dependent-protein kinase catalytic subunit (DNA-PKCS) to repair DNA double strand breaks (DSBs) through the nonhomologous end joining (NHEJ) pathway. As expected mutating these genes in mice caused a similar DSB repair-defective phenotype. However, ku70(-/-) cells and ku80(-/-) cells also appeared to have a defect in base excision repair (BER). BER corrects base lesions, apurinic/apyrimidinic (AP) sites and single stand breaks (SSBs) utilizing a variety of proteins including glycosylases, AP endonuclease 1 (APE1) and DNA Polymerase ß (Pol ß). In addition, deleting Ku70 was not equivalent to deleting Ku80 in cells and mice. Therefore, we hypothesized that free Ku70 (not bound to Ku80) and/or free Ku80 (not bound to Ku70) possessed activity that influenced BER. To further test this hypothesis we performed two general sets of experiments. The first set showed that deleting either Ku70 or Ku80 caused an NHEJ-independent defect. We found ku80(-/-) mice had a shorter life span than dna-pkcs(-/-) mice demonstrating a phenotype that was greater than deleting the holoenzyme. We also found Ku70-deletion induced a p53 response that reduced the level of small mutations in the brain suggesting defective BER. We further confirmed that Ku80-deletion impaired BER via a mechanism that was not epistatic to Pol ß. The second set of experiments showed that free Ku70 and free Ku80 could influence BER. We observed that deletion of either Ku70 or Ku80, but not both, increased sensitivity of cells to CRT0044876 (CRT), an agent that interferes with APE1. In addition, free Ku70 and free Ku80 bound to AP sites and in the case of Ku70 inhibited APE1 activity. These observations support a novel role for free Ku70 and free Ku80 in altering BER.

The mechanism of human tyrosyl-DNA phosphodiesterase 1 in the cleavage of AP site and its synthetic analogs.

The mechanism of hydrolysis of the apurinic/apyrimidinic (AP) site and its synthetic analogs by using tyrosyl-DNA phosphodiesterase 1 (Tdp1) was analyzed. Tdp1 catalyzes the cleavage of AP site and the synthetic analog of the AP site, 3-hydroxy-2(hydroxymethyl)-tetrahydrofuran (THF), in DNA by hydrolysis of the phosphodiester bond between the substituent and 5' adjacent phosphate. The product of Tdp1 cleavage in the case of the AP site is unstable and is hydrolyzed with the formation of 3'- and 5'-margin phosphates. The following repair demands the ordered action of polynucleotide kinase phosphorylase, with XRCC1, DNA polymerase ß, and DNA ligase. In the case of THF, Tdp1 generates break with the 5'-THF and the 3'-phosphate termini. Tdp1 is also able to effectively cleave non-nucleotide insertions in DNA, decanediol and diethyleneglycol moieties by the same mechanism as in the case of THF cleavage. The efficiency of Tdp1 catalyzed hydrolysis of AP-site analog correlates with the DNA helix distortion induced by the substituent. The following repair of 5'-THF and other AP-site analogs can be processed by the long-patch base excision repair pathway.

PrimPol, an archaic primase/polymerase operating in human cells.

We describe a second primase in human cells, PrimPol, which has the ability to start DNA chains with deoxynucleotides unlike regular primases, which use exclusively ribonucleotides. Moreover, PrimPol is also a DNA polymerase tailored to bypass the most common oxidative lesions in DNA, such as abasic sites and 8-oxoguanine. Subcellular fractionation and immunodetection studies indicated that PrimPol is present in both nuclear and mitochondrial DNA compartments. PrimPol activity is detectable in mitochondrial lysates from human and mouse cells but is absent from mitochondria derived from PRIMPOL knockout mice. PRIMPOL gene silencing or ablation in human and mouse cells impaired mitochondrial DNA replication. On the basis of the synergy observed with replicative DNA polymerases Polγ and Polε, PrimPol is proposed to facilitate replication fork progression by acting as a translesion DNA polymerase or as a specific DNA primase reinitiating downstream of lesions that block synthesis during both mitochondrial and nuclear DNA replication.

Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3'-OH base mispairs and damaged base lesions.

T4 RNA ligase 2 (Rnl2) repairs 3'-OH/5'-PO4 nicks in duplex nucleic acids in which the broken 3'-OH strand is RNA. Ligation entails three chemical steps: reaction of Rnl2 with ATP to form a covalent Rnl2-(lysyl-Nζ)-AMP intermediate (step 1); transfer of AMP to the 5'-PO4 of the nick to form an activated AppN- intermediate (step 2); and attack by the nick 3'-OH on the AppN- strand to form a 3'-5' phosphodiester (step 3). Here we used rapid mix-quench methods to analyze the kinetic mechanism and fidelity of single-turnover nick sealing by Rnl2-AMP. For substrates with correctly base-paired 3'-OH nick termini, kstep2 was fast (9.5 to 17.9 sec(-1)) and similar in magnitude to kstep3 (7.9 to 32 sec(-1)). Rnl2 fidelity was enforced mainly at the level of step 2 catalysis, whereby 3'-OH base mispairs and oxoguanine, oxoadenine, or abasic lesions opposite the nick 3'-OH elicited severe decrements in the rate of 5'-adenylylation and relatively modest slowing of the rate of phosphodiester synthesis. The exception was the noncanonical A:oxoG base pair, which Rnl2 accepted as a correctly paired end for rapid sealing. These results underscore (1) how Rnl2 requires proper positioning of the 3'-terminal ribonucleoside at the nick for optimal 5'-adenylylation and (2) the potential for nick-sealing ligases to embed mutations during the repair of oxidative damage.

DNA-N-glycosylases process novel O-glycosidic sites in DNA.

After the hydrolysis of the N-glycosyl bond between a damaged base and C1' of a deoxyribosyl moiety of DNA, human alkyladenine DNA glycosylase (AAG) and Escherichia coli 3-methyladenine DNA glycosylase II (AlkA) bind tightly to their abasic DNA products, potentially protecting these reactive species. Here we show that both AAG and AlkA catalyze reactions between bound abasic DNA and small, primary alcohols to form novel DNA-O-glycosides. The synthesis reactions are reversible, as the DNA-O-glycosides are converted back into abasic DNA upon being incubated with AAG or AlkA in the absence of alcohol. AAG and AlkA are therefore able to hydrolyze O-glycosidic bonds in addition to N-glycosyl bonds. The newly discovered DNA-O-glycosidase activities of both enzymes compare favorably with their known DNA-N-glycosylase activities: AAG removes both methanol and 1,N(6)-ethenoadenine (εA) from DNA with single-turnover rate constants that are 2.9 × 10(5)-fold greater than the corresponding uncatalyzed rates, whereas the rate enhancement of 3.7 × 10(7) for removal of methanol from DNA by AlkA is 300-fold greater than its rate enhancement for removal of εA from DNA. Although the biological significance of the DNA-O-glycosidase reactions is not known, the evolution of new DNA repair pathways may be aided by enzymes that practice catalytic promiscuity, such as these two unrelated DNA glycosylases.