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 I²⁶¹ of Myh1 reduces its interaction with Hus1, but only slightly attenuates its interaction with Apn2. However, E²⁶² 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.     

Action of multiple base excision repair enzymes on the 2'-deoxyribonolactone

Free radical attack on the sugar-phosphate backbone generates oxidized apurinic/apyrimidinic (AP) residues in DNA. 2'-deoxyribonolactone (dL) is a C1'-oxidized AP site damage generated by UV and gamma-irradiation, and certain anticancer drugs. If not repaired dL produces G-->A transitions in Escherichia coli. In the base excision repair (BER) pathway, AP endonucleases are the major enzymes responsible for 5'-incision of the regular AP site (dR) and dL. DNA glycosylases with associated AP lyase activity can also efficiently cleave regular AP sites. Here, we report that dL is a substrate for AP endonucleases but not for DNA glycosylases/AP lyases. The kinetic parameters of the dL-incision were similar to those of the dR. DNA glycosylases such as E. coli formamidopyrimidine-DNA glycosylase, mismatch-specific uracil-DNA glycosylase, and human alkylpurine-DNA N-glycosylase bind strongly to dL without cleaving it. We show that dL cross-links with the human proteins 8-oxoguanine-DNA (hOGG1) and thymine glycol-DNA glycosylases (hNth1), and dR cross-links with Nth and hNth1. These results suggest that dL and dR induced genotoxicity might be strengthened by BER pathway in vivo.     

Mono- and Bifunctional DNA Glycosylases Involved in Repairing Oxidatively Damaged DNA

DNA repair is a basic biological process providing for the stability and integrity of the genome. Disturbed repair results in premature aging, autoimmune and cardiological disorders, tumorigenesis, etc. Data on enzymes which play key roles in repairing DNA with lesions generated by reactive oxygen species are reviewed. The substrate specificity, mechanism of catalysis, structure of the active center, and specific structural and functional features are described for Escherichia coli mono- and bifunctional DNA glycosylases (endonuclease III, Fpg, MutY, endonuclease VIII, AlkA, MutT) and their prokaryotic and eukaryotic homologs (Ntg1, Ntg2, yOgg1, yOgg2, hOgg1, hOgg2, mOgg1, rOgg1, hMTH, hMYH, MAG, ADPG, and ANPG) which are involved in base excision repair.     

Evolution of Base Excision Repair in Entamoeba histolytica is shaped by gene loss,gene duplication,and lateral gene transfer

During its life cycle, the protist parasite Entamoeba histolytica encounters reactive oxygen and nitrogen species that alter its genome. Base excision repair (BER) is one of the most important pathways for the repair of DNA base lesions. Analysis of the E. histolytica genome revealed the presence of most of the BER components. Surprisingly, this included a gene encoding an apurinic/apyrimidinic (AP) endonuclease that previous studies had assumed was absent. Indeed, our analysis showed that the genome of E. histolytica harbors the necessary genes needed for both short and long-patch BER sub-pathways. These genes include DNA polymerases with predicted 5′-dRP lyase and strand-displacement activities and a sole DNA ligase. A distinct feature of the E. histolytica genome is the lack of several key damage-specific BER glycosylases, such as OGG1/MutM, MDB4, Mag1, MPG, SMUG, and TDG. Our evolutionary analysis indicates that several E. histolytica DNA glycosylases were acquired by lateral gene transfer (LGT). The genes that encode for MutY, AlkD, and UDG (Family VI) are included among these cases. Endonuclease III and UNG (family I) are the only DNA glycosylases with a eukaryotic origin in E. histolytica. A gene encoding a MutT 8-oxodGTPase was also identified that was acquired by LGT. The mixed composition of BER genes as a DNA metabolic pathway shaped by LGT in E. histolytica indicates that LGT plays a major role in the evolution of this eukaryote. Sequence and structural prediction of E. histolytica DNA glycosylases, as well as MutT, suggest that the E. histolytica DNA repair proteins evolved to harbor structural modifications that may confer unique biochemical features needed for the biology of this parasite.     

Coordination of MYH DNA glycosylase and APE1 endonuclease activities via physical interactions

MutY homologue (MYH) is a DNA glycosylase which excises adenine paired with the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG, or G^o) during base excision repair (BER). Base excision by MYH results in an apurinic/apyrimidinic (AP) site in the DNA where the DNA sugar–phosphate backbone remains intact. A key feature of MYH activity is its physical interaction and coordination with AP endonuclease I (APE1), which subsequently nicks DNA 5′ to the AP site. Because AP sites are mutagenic and cytotoxic, they must be processed by APE1 immediately after the action of MYH glycosylase. Our recent reports show that the interdomain connector (IDC) of human MYH (hMYH) maintains interactions with hAPE1 and the human checkpoint clamp Rad9–Rad1–Hus1 (9–1–1) complex. In this study, we used NMR chemical shift perturbation experiments to determine hMYH-binding site on hAPE1. Chemical shift perturbations indicate that the hMYH IDC peptide binds to the DNA-binding site of hAPE1 and an additional site which is distal to the APE1 DNA-binding interface. In these two binding sites, N212 and Q137 of hAPE1 are key mediators of the MYH/APE1 interaction. Intriguingly, despite the fact that hHus1 and hAPE1 both interact with the MYH IDC, hHus1 does not compete with hAPE1 for binding to hMYH. Rather, hHus1 stabilizes the hMYH/hAPE1 complex both in vitro and in cells. This is consistent with a common theme in BER, namely that the assembly of protein–DNA complexes enhances repair by efficiently coordinating multiple enzymatic steps while simultaneously minimizing the release of harmful repair intermediates.     

Dynamic Processing of a Common Oxidative DNA Lesion by the First Two Enzymes of the Base Excision Repair Pathway

Base excision repair (BER) is the primary pathway by which eukaryotic cells resolve single base damage. One common example of single base damage is 8-oxo-7,8-dihydro-2ʹ-deoxoguanine (8-oxoG). High incidence and mutagenic potential of 8-oxoG necessitate rapid and efficient DNA repair. How BER enzymes coordinate their activities to resolve 8-oxoG damage while limiting cytotoxic BER intermediates from propagating genomic instability remains unclear. Here we use single-molecule Förster resonance energy transfer (smFRET) and ensemble-level techniques to characterize the activities and interactions of consecutive BER enzymes important for repair of 8-oxoG. In addition to characterizing the damage searching and processing mechanisms of human 8-oxoguanine glycosylase 1 (hOGG1), our data support the existence of a ternary complex between hOGG1, the damaged DNA substrate, and human AP endonuclease 1 (APE1). Our results indicate that hOGG1 is actively displaced from its abasic site containing product by protein–protein interactions with APE1 to ensure timely repair of damaged DNA.     

Arabidopsis ARP endonuclease functions in a branched base excision DNA repair pathway completed by LIG1

Base excision repair (BER) is an essential cellular defence mechanism against DNA damage, but it is poorly understood in plants. We used an assay that monitors repair of damaged bases and abasic (apurinic/apyrimidinic, AP) sites in Arabidopsis to characterize post-excision events during plant BER. We found that Apurinic endonuclease-redox protein (ARP) is the major AP endonuclease activity in Arabidopsis cell extracts, and is required for AP incision during uracil BER in vitro. Mutant plants that are deficient in ARP grow normally but are hypersensitive to 5-fluorouracil, a compound that favours mis-incorporation of uracil into DNA. We also found that, after AP incision, the choice between single-nucleotide or long-patch DNA synthesis (SN- or LP-BER) is influenced by the 5' end of the repair gap. When the 5' end is blocked and not amenable to β-elimination, the SN sub-pathway is abrogated, and repair is accomplished through LP-BER only. Finally, we provide evidence that Arabidopsis DNA ligase I (LIG1) is required for both SN- and LP-BER. lig1 RNAi-silenced lines show very reduced uracil BER, and anti-LIG1 antibody abolishes repair in wild-type cell extracts. In contrast, knockout lig4(-/-) mutants exhibit normal BER and nick ligation levels. Our results suggest that a branched BER pathway completed by a member of the DNA ligase I family may be an ancient feature in eukaryotic species.     

Single‐nucleotide and long‐patch base excision repair of DNA damage in plants

Base excision repair (BER) is a critical pathway in cellular defense against endogenous or exogenous DNA damage. This elaborate multistep process is initiated by DNA glycosylases that excise the damaged base, and continues through the concerted action of additional proteins that finally restore DNA to the unmodified state. BER has been subject to detailed biochemical analysis in bacteria, yeast and animals, mainly through in vitro reproduction of the entire repair reaction in cell‐free extracts. However, an understanding of this repair pathway in plants has consistently lagged behind. We report the extension of BER biochemical analysis to plants, using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro. We have used this system to demonstrate that Arabidopsis cell extracts contain the enzymatic machinery required to completely repair ubiquitous DNA lesions, such as uracil and abasic (AP) sites. Our results reveal that AP sites generated after uracil excision are processed both by AP endonucleases and AP lyases, generating either 5′‐ or 3′‐blocked ends, respectively. We have also found that gap filling and ligation may proceed either through insertion of just one nucleotide (short‐patch BER) or several nucleotides (long‐patch BER). This experimental system should prove useful in the biochemical and genetic dissection of BER in plants, and contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways.     

Modulation of oxidative mutagenesis and carcinogenesis by polymorphic forms of human DNA repair enzymes

Chromosome DNA is continuously exposed to various endogenous and exogenous mutagens. Among them, oxidation is one of the most common threats to genetic stability, and multiple DNA repair enzymes protect chromosome DNA from the oxidative damage. In Escherichia coli, three repair enzymes synergistically reduce the mutagenicity of oxidized base 8-hydroxy-guanine (8-OH-G). MutM DNA glycosylase excises 8-OH-G from 8-OH-G:C pairs in DNA and MutY DNA glycosylase removes adenine incorporated opposite template 8-OH-G during DNA replication. MutT hydrolyzes 8-OH-dGTP to 8-OH-dGMP in dNTP pool, thereby reducing the chance of misincorporation of 8-OH-dGTP by DNA polymerases. Simultaneous inactivation of MutM and MutY dramatically increases the frequency of spontaneous G:C to T:A mutations, and the deficiency of MutT leads to the enhancement of T:A to G:C transversions more than 1000-fold over the control level. In humans, the functional homologues of MutM, MutY and MutT, i.e., OGG1, MUTYH (MYH) and MTH1, contribute to the protection of genomic DNA from oxidative stress. Interestingly, several polymorphic forms of these proteins exist in human populations, and some of them are suggested to be associated with cancer susceptibility. Here, we review the polymorphic forms of OGG1, MUTYH and MTH1 involved in repair of 8-OH-G and 8-OH-dGTP, and discuss the significance of the polymorphisms in the maintenance of genomic integrity. We also summarize the polymorphic forms of human DNA polymerase eta, which may be involved in damage tolerance and mutagenesis induced by oxidative stress.     

Transient protein–protein complexes in base excision repair

Abstract

Transient protein–protein complexes are of great importance for organizing multiple enzymatic reactions into productive reaction pathways. Base excision repair (BER), a process of critical importance for maintaining genome stability against a plethora of DNA-damaging factors, involves several enzymes, including DNA glycosylases, AP endonucleases, DNA polymerases, DNA ligases and accessory proteins acting sequentially on the same damaged site in DNA. Rather than being assembled into one stable multisubunit complex, these enzymes pass the repair intermediates between them in a highly coordinated manner. In this review, we discuss the nature and the role of transient complexes arising during BER as deduced from structural and kinetic data. Almost all of the transient complexes are DNA-mediated, although some may also exist in solution and strengthen under specific conditions. The best-studied example, the interactions between DNA glycosylases and AP endonucleases, is discussed in more detail to provide a framework for distinguishing between stable and transient complexes based on the kinetic data.

Communicated by Ramaswamy H. Sarma     

Purification and characterization of a mammalian homolog of Escherichia coli MutY mismatch repair protein from calf liver mitochondria

A protein homologous to the Escherichia coli MutY glycosylase, referred to as mtMYH, has been purified from calf liver mitochondria. SDS–polyacrylamide gel electrophoresis, western blot analysis as well as gel filtration chromatography predicted the molecular mass of the purified calf mtMYH to be 35–40 kDa. Gel mobility shift analysis showed that the purified mtMYH formed specific binding complexes with A/8-oxoG, G/8-oxoG and T/8-oxoG, weakly with C/8-oxoG, but not with A/G and A/C mismatches. The purified mtMYH exhibited DNA glycosylase activity removing adenine mispaired with G, C or 8-oxoG and weakly removing guanine mispaired with 8-oxoG. The mtMYH glycosylase activity was insensitive to high concentrations of NaCl and EDTA. The purified mtMYH cross-reacted with antibodies against both intact MutY and a peptide of human MutY homolog (hMYH). DNA glycosylase activity of mtMYH was inhibited by anti-MutY antibodies but not by anti-hMYH peptide antibodies. Together with the previously described mitochondrial MutT homolog (MTH1) and 8-oxoG glycosylase (OGG1, a functional MutM homolog), mtMYH can protect mitochondrial DNA from the mutagenic effects of 8-oxoG.     

Recognition of the oxidized lesions spiroiminodihydantoin and guanidinohydantoin in DNA by the mammalian base excision repair glycosylases NEIL1 and NEIL2

8-Oxoguanine (8-oxoG) is an unstable mutagenic DNA lesion that is prone to further oxidation. High valent metals such as Cr(V) and Ir(IV) readily oxidize 8-oxoG to form guanidinohydantoin (Gh), its isomer iminoallantoin (Ia), and spiroiminodihydantoin (Sp). When present in DNA, these lesions show enhanced base misincorporation over the parent 8-oxoG lesion leading to G --> T and G --> C transversion mutations and polymerase arrest. These findings suggested that further oxidized lesions of 8-oxoG are more mutagenic and toxic than 8-oxoG itself. Repair of oxidatively damaged bases, including Sp and Gh/Ia, are initiated by the base excision repair (BER) system that involves the DNA glycosylases Fpg, Nei, and Nth in E. coli. Mammalian homologs of two of these BER enzymes, OGG1 and NTH1, have little or no affinity for Gh/Ia and Sp. Herein we report that two recently identified mammalian glycosylases, NEIL1 and NEIL2, showed a high affinity for recognition and cleavage of DNA containing Gh/Ia and Sp lesions. NEIL1 and NEIL2 recognized both of these lesions in single-stranded DNA and catalyzed the removal of the lesions through a beta- and delta-elimination mechanism. NEIL1 and NEIL2 also recognized and excised the Gh/Ia lesion opposite all four natural bases in double-stranded DNA. NEIL1 was able to excise the Sp lesion opposite the four natural bases in double-stranded DNA, however, NEIL2 showed little cleavage activity against the Sp lesion in duplex DNA although DNA trapping studies show recognition and binding of NEIL2 to this lesion. This work suggests that NEIL1 and NEIL2 are essential in the recognition of further oxidized lesions arising from 8-oxoG and implies that these BER glycosylases may play an important role in the repair of DNA damage induced by carcinogenic metals.     

Substrate specificities and excision kinetics of DNA glycosylases involved in base-excision repair of oxidative DNA damage

Reactive oxygen-derived species such as free radicals are formed in living cells by normal metabolism and exogenous sources, and cause a variety of types of DNA damage such as base and sugar damage, strand breaks and DNA-protein cross-links. Living organisms possess repair systems that repair DNA damage. Oxidative DNA damage caused by free radicals and other oxidizing agents is mainly repaired by base-excision repair (BER), which involves DNA glycosylases in the first step of the repair process. These enzymes remove modified bases from DNA by hydrolyzing the glycosidic bond between the modified base and the sugar moiety, generating an apurinic/apyrimidinic (AP) site. Some also possess AP lyase activity that subsequently cleaves DNA at AP sites. Many DNA glycosylases have been discovered and isolated, and their reaction mechanisms and substrate specificities have been elucidated. Most of the known products of oxidative damage to DNA are substrates of DNA glycosylases with broad or narrow substrate specificities. Some possess cross-activity and remove both pyrimidine- and purine-derived lesions. Overlapping activities between enzymes also exist. Studies of substrate specificities have been performed using either oligodeoxynucleotides with a single modified base embedded at a specific position or damaged DNA substrates containing a multiplicity of pyrimidine- and purine-derived lesions. This paper reviews the substrate specificities and excision kinetics of DNA glycosylases that have been investigated with the use of gas chromatography/mass spectrometry and DNA substrates with multiple lesions.     

Effect of the multifunctional proteins RPA, YB-1, and XPC repair factor on AP site cleavage by DNA glycosylase NEIL1

DNA glycosylases are key enzymes in the first step of base excision DNA repair, recognizing DNA damage and catalyzing the release of damaged nucleobases. Bifunctional DNA glycosylases also possess associated apurinic/apyrimidinic (AP) lyase activity that nick the damaged DNA strand at an abasic (or AP) site, formed either spontaneously or at the first step of repair. NEIL1 is a bifunctional DNA glycosylase capable of processing lesions, including AP sites, not only in double-stranded but also in single-stranded DNA. Here, we show that proteins participating in DNA damage response, YB-1 and RPA, affect AP site cleavage by NEIL1. Stimulation of the AP lyase activity of NEIL1 was observed when an AP site was located in a 60 nt-long double-stranded DNA. Both RPA and YB-1 inhibited AP site cleavage by NEIL1 when the AP site was located in single-stranded DNA. Taking into account a direct interaction of YB-1 with the AP site, located in single-stranded DNA, and the high affinity of both YB-1 and RPA for single-stranded DNA, this behavior is presumably a consequence of a competition with NEIL1 for the DNA substrate. Xeroderma pigmentosum complementation group C protein (XPC), a key protein of another DNA repair pathway, was shown to interact directly with AP sites but had no effect on AP site cleavage by NEIL1.     

A single engineered point mutation in the adenine glycosylase MutY confers bifunctional glycosylase/AP lyase activity

The E. coli adenine glycosylase MutY is a member of the base excision repair (BER) superfamily of DNA repair enzymes. MutY plays an important role in preventing mutations caused by 7, 8-dihydro-8-oxo-2'-deoxyguanosine (OG) by removing adenine from OG:A base pairs. Some enzymes of the BER superfamily catalyze a strand scission even concomitant with base removal. These bifunctional glycosylase/AP lyases bear a conserved lysine group in the active site region, which is believed to be the species performing the initial nucleophilic attack at C1' in the catalysis of base removal. Monofunctional glycosylases such as MutY are thought to perform this C1' nucleophilic displacement by a base-activated water molecule, and, indeed, the conservation of amine functionality positioning has not been observed in protein sequence alignments. Bifunctional glycosylase/AP lyase activity was successfully engineered into MutY by replacing serine 120 with lysine. MutY S120K is capable of catalyzing DNA strand scission at a rate equivalent to that of adenine excision for both G:A and OG:A mispair substrates. The extent of DNA backbone cleavage is independent of treating reaction aliquots with 0.1 M NaOH. Importantly, the replacement of the serine with lysine results in a catalytic rate that is compromised by at least 20-fold. The reduced efficiency in the glycosylase activity is also reflected in a reduced ability of S120K MutY to prevent DNA mutations in vivo. These results illustrate that the mechanisms of action of the two classes of these enzymes are quite similar, such that a single amino acid change is sufficient, in the case of MutY, to convert a monofunctional glycosylase to a bifunctional glycosylase/AP lyase.     

Base excision repair activities required for yeast to attain a full chronological life span

The chronological life span of yeast, the survival of stationary (G0) cells over time, provides a model for investigating certain of the factors that may influence the aging of non-dividing cells and tissues in higher organisms. This study measured the effects of defined defects in the base excision repair (BER) system for DNA repair on this life span. Stationary yeast survives longer when it is pre-grown on respiratory, as compared to fermentative (glucose), media. It is also less susceptible to viability loss as the result of defects in DNA glycosylase/AP lyases (Ogg1p, Ntg1p, Ntg2p), apurinic/apyrimidinic (AP) endonucleases (Apn1p, Apn2p) and monofunctional DNA glycosylase (Mag1p). Whereas single BER glycosylase/AP lyase defects exerted little influence over such optimized G0 survival, this survival was severely shortened with the loss of two or more such enzymes. Equally, the apn1delta and apn2delta single gene deletes survived as well as the wild type, whereas a apn1delta apn2delta double mutant totally lacking in any AP endonuclease activity survived poorly. Both this shortened G0 survival and the enhanced mutagenicity of apn1delta apn2delta cells were however rescued by the over-expression of either Apn1p or Apn2p. The results highlight the vital importance of BER in the prevention of mutation accumulation and the attainment of the full yeast chronological life span. They also reveal an appreciable overlap in the G0 maintenance functions of the different BER DNA glycosylases and AP endonucleases.     

Recent progress in the biology, chemistry and structural biology of DNA glycosylases

Since the discovery in 1974 of uracil DNA glycosylase (UDG), the first member of the family of enzymes involved in base excision repair (BER), considerable progress has been made in the understanding of DNA glycosylases, the polypeptides that remove damaged or mispaired DNA bases from DNA. We also know the enzymes that act downstream of the glycosylases, in the processing of abasic sites, in gap filling and in DNA ligation. This article covers the most recent developments in our understanding of BER, with particular emphasis on the mechanistic aspects of this process, which have been made possible by the elucidation of the crystal structures of several glycosylases in complex with their respective substrates, substrate analogues and products. The biological importance of individual BER pathways is also being appreciated through the inactivation of key BER genes in knockout mouse models.     

Arabidopsis ZDP DNA 3′‐phosphatase and ARP endonuclease function in 8‐oxoG repair initiated by FPG and OGG1 DNA glycosylases

Oxidation of guanine in DNA generates 7,8‐dihydro‐8‐oxoguanine (8‐oxoG), an ubiquitous lesion with mutagenic properties. 8‐oxoG is primarily removed by DNA glycosylases distributed in two families, typified by bacterial Fpg proteins and eukaryotic Ogg1 proteins. Interestingly, plants possess both Fpg and Ogg1 homologs but their relative contributions to 8‐oxoG repair remain uncertain. In this work we used Arabidopsis cell‐free extracts to monitor 8‐oxoG repair in wild‐type and mutant plants. We found that both FPG and OGG1 catalyze excision of 8‐oxoG in Arabidopsis cell extracts by a DNA glycosylase/lyase mechanism, and generate repair intermediates with blocked 3′‐termini. An increase in oxidative damage is detected in both nuclear and mitochondrial DNA from double fpg ogg1 mutants, but not in single mutants, which suggests that a single deficiency in one of these DNA glycosylases may be compensated by the other. We also found that the DNA 3′‐phosphatase ZDP (zinc finger DNA 3′‐phosphoesterase) and the AP(apurinic/apyirmidinic) endonuclease ARP(apurinic endonuclease redox protein) are required in the 8‐oxoG repair pathway to process the 3′‐blocking ends generated by FPG and OGG1. Furthermore, deficiencies in ZDP and/or ARP decrease germination ability after seed deteriorating conditions. Altogether, our results suggest that Arabidopsis cells use both FPG and OGG1 to repair 8‐oxoG in a pathway that requires ZDP and ARP in downstream steps.     

A general role of the DNA glycosylase Nth1 in the abasic sites cleavage step of base excision repair in Schizosaccharomyces pombe

One of the most frequent lesions formed in cellular DNA are abasic (apurinic/apyrimidinic, AP) sites that are both cytotoxic and mutagenic, and must be removed efficiently to maintain genetic stability. It is generally believed that the repair of AP sites is initiated by the AP endonucleases; however, an alternative pathway seems to prevail in Schizosaccharomyces pombe. A mutant lacking the DNA glycosylase/AP lyase Nth1 is very sensitive to the alkylating agent methyl methanesulfonate (MMS), suggesting a role for Nth1 in base excision repair (BER) of alkylation damage. Here, we have further evaluated the role of Nth1 and the second putative S.pombe AP endonuclease Apn2, in abasic site repair. The deletion of the apn2 open reading frame dramatically increased the sensitivity of the yeast cells to MMS, also demonstrating that the Apn2 has an important function in the BER pathway. The deletion of nth1 in the apn2 mutant strain partially relieves the MMS sensitivity of the apn2 single mutant, indicating that the Apn2 and Nth1 act in the same pathway for the repair of abasic sites. Analysis of the AP site cleavage in whole cell extracts of wild-type and mutant strains showed that the AP lyase activity of Nth1 represents the major AP site incision activity in vitro. Assays with DNA substrates containing base lesions removed by monofunctional DNA glycosylases Udg and MutY showed that Nth1 will also cleave the abasic sites formed by these enzymes and thus act downstream of these enzymes in the BER pathway. We suggest that the main function of Apn2 in BER is to remove the resulting 3′-blocking termini following AP lyase cleavage by Nth1.