In nucleotide incision repair (NIR), an endonuclease nicks oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to the repair of the remaining 5'-dangling modified nucleotide. This mechanistic feature is distinct from DNA glycosylase-mediated base excision repair. Here we report that Ape1, the major apurinic/apyrimidinic endonuclease in human cells, is the damage- specific endonuclease involved in NIR. We show that Ape1 incises DNA containing 5,6-dihydro-2'-deoxyuridine, 5,6-dihydrothymidine, 5-hydroxy-2'-deoxyuridine, alpha-2'-deoxyadenosine and alpha-thymidine adducts, generating 3'-hydroxyl and 5'-phosphate termini. The kinetic constants indicate that Ape1-catalysed NIR activity is highly efficient. The substrate specificity and protein conformation of Ape1 is modulated by MgCl2 concentrations, thus providing conditions under which NIR becomes a major activity in cell-free extracts. While the N-terminal region of Ape1 is not required for AP endonuclease function, we show that it regulates the NIR activity. The physiological relevance of the mammalian NIR pathway is discussed.

ABSrRACTThymine glycols were produced in M13 DNA in a concentration dependent manner by treating the DNA with osmium tetroxide (0s04). For the formation of urea-containing M13 DNA, OsO 4-oxidized DNA was hydrolyzed in alkali (pH 12) to convert the thymine glycols to urea residues. With both thymine glycol-and urea-containing M13 DNA, DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment was decreased in proportion to the number of damages present in the template DNA. Sequencing gel analysis of the products synthesized by E. coli DNA polymerase I and T4 DNA polymerase showed that DNA synthesis terminated opposite the putative thymine glycol site and at one nucleotide before the putative urea site. Substitution of manganese for magnesium in the reaction mix resulted in increased processivity of DNA synthesis so that a base was incorporated opposite urea. With thymine glycol-containing DNA, processivity in the presence of manganese was strongly dependent on the presence of a pyrimidine 5' to the thymine glycol in the template.

DNA-protein crosslinks (DPCs)-where proteins are covalently trapped on the DNA strand-block the progression of replication and transcription machineries and hence hamper the faithful transfer of genetic information. However, the repair mechanism of DPCs remains largely elusive. Here we have analyzed the roles of nucleotide excision repair (NER) and homologous recombination (HR) in the repair of DPCs both in vitro and in vivo using a bacterial system. Several lines of biochemical and genetic evidence show that both NER and HR commit to the repair or tolerance of DPCs, but differentially. NER repairs DPCs with crosslinked proteins of sizes less than 12-14 kDa, whereas oversized DPCs are processed exclusively by RecBCD-dependent HR. These results highlight how NER and HR are coordinated when cells need to deal with unusually bulky DNA lesions such as DPCs.

DNA-protein cross-links (DPCsaccount for a class of the most ubiquitous DNA lesions and are known to be produced by chemical agents, such as formaldehyde (FA) and transition metals, and by physical agents, such as ionizing radiation and UV light (1). DPCs are also produced by anticancer drugs, such as 5-aza-2Ј-deoxycytidine (azadC) and cisplatin (1, 2). Although some classes of DPCs contain a flanking strand break (e.g. covalently trapped topoisomerases) (3), typical (and probably the most common) DPCs contain proteins irreversibly trapped on the uninterrupted DNA strand. It is readily inferred from the unusually bulky nature of crosslinked proteins (CLPs) that steric hindrance imposed by CLPs on proteins involved in DNA transactions would hamper replication, transcription, and repair. Consistent with this notion, DPCs incorporated into oligonucleotides and plasmid DNA block DNA replication in vitro (4, 5) and in vivo (6, 7), respectively. Moreover, CLPs attenuate the binding of the damage recognition protein (UvrB) involved in bacterial nucleotide excision repair (NER) in a size-dependent manner (7).Conversely, it has been largely elusive how cells circumvent the genotoxic effects of DPCs. We recently showed that NER and homologous recombination (HR) play pivotal roles in mitigating the genotoxic effects of DPCs in bacteria (7). Interestingly, the two pathways contribute differentially to the tolerance of DPCs. In NER catalyzed by UvrABC, the excision efficiency for DPCs varies dramatically with the size of CLPs both in vitro and in vivo and is attenuated by steric hindrance of CLPs. The upper size limit of CLPs amenable to NER in vitro was around 16 kDa, but the biologically relevant size limit was lower in vivo, at around 11 kDa. DPCs with oversized CLPs are processed exclusively by RecB-dependent HR. Given that HR * This work was supported in part by Grants-in-aid for Scientific Research from the Japan Society for the Promotion of Science (to T. N., H. T., and H. I.) and by a Grant-in-aid for the Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology (to H. I.

Free radicals produce a wide spectrum of damages; among these are DNA base damages and abasic (AP) sites. Although several methods have been used to detect and quantify AP sites, they either are relatively laborious or require the use of radioactivity. A novel reagent for detecting abasic sites in DNA was prepared by reacting O-(carboxymethyl)hydroxylamine with biotin hydrazide in the presence of carbodiimide. This reagent, called Aldehyde Reactive Probe (ARP), specifically tagged AP sites in DNA with biotin residues. The number of biotin-tagged AP sites was then determined colorimetrically by an ELISA-like assay using avidin/biotin complex conjugated to horseradish peroxidase as the indicator enzyme. With heat/acid-depurinated calf thymus or bacteriophage f1 DNA, ARP detected femtomoles of AP sites in DNA. Using this assay, DNA damages generated in calf thymus, phi X174 RF, and f1 single-stranded DNA, X-irradiated in phosphate buffer, were easily detectable at 10 rad (0.1 Gy). Furthermore, ARP sites were detectable in DNA isolated from heat-inactivated X-irradiated (10 Gy) and methyl methanesulfonate (MMS)-treated (5 microM) Escherichia coli cells. The rate of production of ARP sites was proportional to the X-ray dose as well as to the concentration of MMS. Thus, the sensitivity and simplicity of the ARP assay should provide a potentially powerful method for the quantitation of AP sites or other DNA lesions containing an aldehyde group.

No abstract

Endonuclease VIII, a novel presumptive DNA repair enzyme, was isolated from Escherichia coli by FPLC1 purification. The enzyme was found in strains that contained or lacked endonuclease III and was purified by radial flow S-Sepharose, Mono S, phenyl-Superose, and Superose 12 FPLC. Examination of the properties of endonuclease VIII showed it to have many similarities to endonuclease III. DNA containing thymine glycol, dihydrothymine, beta-ureidoisobutyric acid, urea residues, or AP sites was incised by the enzyme; however, DNA containing reduced AP sites was not. HPLC analysis of the products formed by exhaustive enzymatic digestion of damage-containing DNA showed that endonuclease VIII released thymine glycol and dihydrothymine as free bases. Taken together, these data suggest that endonuclease VIII contains both N-glycosylase and AP lyase activities. Consistent with this idea, DNA containing AP sites or thymine glycols, that was enzymatically nicked by endonuclease VIII was not a good substrate for E. coli DNA polymerase I, suggesting that endonuclease VIII nicks damage-containing DNA on the 3' side of the lesion. Also, since monophosphates were not released after treating thymine glycol-containing DNA with endonuclease VIII, the enzyme does not appear to have exonuclease activity. The enzyme activity was maximal in 75 mM NaCl or 5 mM MgCl2. Analysis of endonuclease VIII by both Superose FPLC and Sephadex yielded native molecular masses of 28,000 and 30,000 Da, respectively. SDS-PAGE, in conjunction with activity gel analysis, gave a molecular mass of about 29,000 Da. Furthermore, renaturation of the putative active band from SDS-PAGE gave rise to an active enzyme.

In human cells, oxidative pyrimidine lesions are restored by the base excision repair pathway initiated by homologues of Endo III (hNTH1) and Endo VIII (hNEIL1 and hNEIL2). In this study we have quantitatively analyzed and compared their activity toward nine oxidative base lesions and an apurinic/apyrimidinic (AP) site using defined oligonucleotide substrates. hNTH1 and hNEIL1 but not hNEIL2 excised the two stereoisomers of thymine glycol (5R-Tg and 5S-Tg), but their isomer specificity was markedly different: the relative activity for 5R-Tg:5S-Tg was 13:1 for hNTH1 and 1.5:1 for hNEIL1. This was also the case for their Escherichia coli homologues: the relative activity for 5R-Tg:5S-Tg was 1:2.5 for Endo III and 3.2:1 for Endo VIII. Among other tested lesions for hNTH1, an AP site was a significantly better substrate than urea, 5-hydroxyuracil (hoU), and guanine-derived formamidopyrimidine (mFapyG), whereas for hNEIL1 these base lesions and an AP site were comparable substrates. In contrast, hNEIL2 recognized an AP site exclusively, and the activity for hoU and mFapyG was marginal. hNEIL1, hNEIL2, and Endo VIII but not hNTH1 and Endo III formed cross-links to oxanine, suggesting conservation of the -fold of the active site of the Endo VIII homologues. The profiles of the excision of the Tg isomers with HeLa and E. coli cell extracts closely resembled those of hNTH1 and Endo III, confirming their major contribution to the repair of Tg isomers in cells. However, detailed analysis of the cellular activity suggests that hNEIL1 has a significant role in the repair of 5S-Tg in human cells.DNA carrying vital genetic information of cells constantly suffers from spontaneous deamination and depurination, alkylation, and oxidation (1-3). These reactions lead to modifications of the DNA backbone and bases, with the latter predominating. The resulting aberrant bases are potentially genotoxic because of the loss or alteration of base pairing information (4), and hence need to be restored by the cellular repair system (2, 3, 5). The major repair mechanism for such damage is the base excision repair (BER) 1 pathway (6), which is conserved from bacteria to humans. In the first step of BER, DNA glycosylases with distinct damage specificities detect the aberrant base in the vast sea of normal bases and remove it from the DNA backbone, leaving an apurinic/apyrimidinic (AP) site. The resulting AP site is further processed and repaired by the subsequent action of AP endonuclease (Endo), DNA polymerase, and DNA ligase through the short patch or long patch BER pathway.The initial search for DNA glycosylases involved in the repair of oxidatively damaged bases in Escherichia coli identified Endo III, Endo VIII, and formamidopyrimidine-DNA glycosylase (7,8). The principal substrates of Endo III and Endo VIII are oxidative pyrimidine lesions. They exhibit redundant damage specificity and catalyze the hydrolysis of the N-glycosidic bond (N-glycosylase activity) and the subsequent incision of an AP site by AP lyase activity via ␤-elimination ...