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.

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 ...

SummaryUpon blockage of chromosomal replication by DNA lesions, Y-family polymerases interact with monoubiquitylated proliferating cell nuclear antigen (PCNA) to catalyse translesion synthesis (TLS) and restore replication fork progression. Here, we assessed the roles of Arabidopsis thaliana POLH, which encodes a homologue of Y-family polymerase g (Polg), PCNA1 and PCNA2 in TLS-mediated UV resistance. A T-DNA insertion in POLH sensitized the growth of roots and whole plants to UV radiation, indicating that AtPolg contributes to UV resistance. POLH alone did not complement the UV sensitivity conferred by deletion of yeast RAD30, which encodes Polg, although AtPolg exhibited cyclobutane dimer bypass activity in vitro, and interacted with yeast PCNA, as well as with Arabidopsis PCNA1 and PCNA2. Co-expression of POLH and PCNA2, but not PCNA1, restored normal UV resistance and mutation kinetics in the rad30 mutant. A single residue difference at site 201, which lies adjacent to the residue (lysine 164) ubiquitylated in PCNA, appeared responsible for the inability of PCNA1 to function with AtPolg in UV-treated yeast. PCNA-interacting protein boxes and an ubiquitin-binding motif in AtPolg were found to be required for the restoration of UV resistance in the rad30 mutant by POLH and PCNA2. These observations indicate that AtPolg can catalyse TLS past UV-induced DNA damage, and links the biological activity of AtPolg in UV-irradiated cells to PCNA2 and PCNA-and ubiquitinbinding motifs in AtPolg.

Nitric oxide (NO) induces deamination of guanine, yielding xanthine and oxanine (Oxa). Furthermore, Oxa reacts with polyamines and DNA binding proteins to form cross-link adducts. Thus, it is of interest how these lesions are processed by DNA repair enzymes in view of the genotoxic mechanism of NO. In the present study, we have examined the repair capacity for Oxa and Oxa–spermine cross-link adducts (Oxa–Sp) of enzymes involved in base excision repair (BER) and nucleotide excision repair (NER) to delineate the repair mechanism of nitrosative damage to guanine. Oligonucleotide substrates containing Oxa and Oxa–Sp were incubated with purified BER and NER enzymes or cell-free extracts (CFEs), and the damage-excising or DNA-incising activity was compared with that for control (physiological) substrates. The Oxa-excising activities of Escherichia coli and human DNA glycosylases and HeLa CFEs were 0.2–9% relative to control substrates, implying poor processing of Oxa by BER. In contrast, DNA containing Oxa–Sp was incised efficiently by UvrABC nuclease and SOS-induced E.coli CFEs, suggesting a role of NER in ameliorating genotoxic effects associated with nitrosative stress. Analyses of the activity of CFEs from NER-proficient and NER-deficient human cells on Oxa–Sp DNA confirmed further the involvement of NER in the repair of nitrosative DNA damage.

Human DNA is continuously damaged by exogenous and endogenous genotoxic insults. To counteract DNA damage and ensure the completion of DNA replication, cells possess specialized DNA polymerases (Pols) that bypass a variety of DNA lesions. Human DNA polymerase kappa (hPolkappa) is a member of the Y-family of DNA Pols and a direct counterpart of DinB in Escherichia coli. hPolkappa is characterized by its ability to bypass several DNA adducts [e.g., benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) and thymine glycol] and efficiently extend primers with mismatches at the termini. hPolkappa is structurally distinct from E. coli DinB in that it possesses an approximately 100-amino acid extension at the N-terminus. Here, we report that tyrosine 112 (Y112), the steric gate amino acid of hPolkappa, which distinguishes dNTPs from rNTPs by sensing the 2'-hydroxy group of incoming nucleotides, plays a crucial role in extension reactions with mismatched primer termini. When Y112 was replaced with alanine, the amino acid change severely reduced the catalytic constant, i.e., k(cat), of the extending mismatched primers and lowered the efficiency, i.e., k(cat)/K(m), of this process by approximately 400-fold compared with that of the wild-type enzyme. In contrast, the amino acid replacement did not reduce the insertion efficiency of dCMP opposite BPDE-N(2)-dG in template DNA, nor did it affect the ability of hPolkappa to bind strongly to template-primer DNA with BPDE-N(2)-dG/dCMP. We conclude that the steric gate of hPolkappa is a major fidelity factor that regulates extension reactions from mismatched primer termini.

Excess risk of leukemia and brain tumors after CT scans in children has been reported. We performed dicentric chromosome assay (DCAs) before and after CT scan to assess effects of low-dose ionizing radiation on chromosomes. Peripheral blood (PB) lymphocytes were collected from 10 patients before and after a CT scan. DCA was performed by analyzing either 1,000 or 2,000 metaphases using both Giemsa staining and centromere-fluorescence in situ hybridization (Centromere-FISH). The increment of DIC formation was compared with effective radiation dose calculated using the computational dosimetry system, WAZA-ARI and dose length product (DLP) in a CT scan. Dicentric chromosome (DIC) formation increased significantly after a single CT scan, and increased DIC formation was found in all patients. A good correlation between the increment of DIC formation determined by analysis of 2,000 metaphases using Giemsa staining and those by 2,000 metaphases using Centromere-FISH was observed. However, no correlation was observed between the increment of DIC formation and the effective radiation dose. Therefore, these results suggest that chromosome cleavage may be induced by one CT scan, and we recommend 2,000 or more metaphases be analyzed in Giemsa staining or Centromere-FISH for DCAs in cases of low-dose radiation exposure.

Oxidized DNA precursors can cause mutagenesis and carcinogenesis when they are incorporated into the genome. Some human Y-family DNA polymerases (Pols) can effectively incorporate 8-oxo-dGTP, an oxidized form of dGTP, into a position opposite a template dA. This inappropriate G:A pairing may lead to transversions of A to C. To gain insight into the mechanisms underlying erroneous nucleotide incorporation, we changed amino acids in human Polη and Polκ proteins that might modulate their specificity for incorporating 8-oxo-dGTP into DNA. We found that Arg61 in Polη was crucial for erroneous nucleotide incorporation. When Arg61 was substituted with lysine (R61K), the ratio of pairing of dA to 8-oxo-dGTP compared to pairing of dC was reduced from 660:1 (wild-type Polη) to 7 : 1 (R61K). Similarly, Tyr112 in Polκ was crucial for erroneous nucleotide incorporation. When Tyr112 was substituted with alanine (Y112A), the ratio of pairing was reduced from 11: 1 (wild-type Polκ) to almost 1: 1 (Y112A). Interestingly, substitution at the corresponding position in Polη, i.e. Phe18 to alanine, did not alter the specificity. These results suggested that amino acids at distinct positions in the active sites of Polη and Polκ might enhance 8-oxo-dGTP to favor the syn conformation, and thus direct its misincorporation into DNA.