Expression of DNA ligase IV is linked to poor prognosis and characterizes a subset of prostate cancers harboring TMPRSS2:ERG fusion and PTEN deletion.

DNA ligases are essential for the maintenance of genome integrity as they are indispensable for DNA replication, recombination and repair. The present study was undertaken to gain insights into the prevalence and clinical significance of ligase IV (LIG4) expression in prostate cancer. A total of 11,152 prostate cancer specimens were analyzed by immunohistochemistry for LIG4 expression. Results were compared to follow-up data, ERG status and deletions at PTEN, 3p13, 5q21 and 6q15. LIG4 expression was predominantly localized in the nucleus of the cells with increased intensities in malignant as compared to benign prostate epithelium. In prostate cancer, LIG4 expression was found in 91% of interpretable tumors, including 12% cancers with weak, 23% with moderate and 56% with strong LIG4 positivity. Strong LIG4 expression was tightly linked to advanced Gleason score (P<0.0001) and positive nodal involvement (P=0.03). There was a remarkable accumulation of strong LIG4 expression in tumors harboring TMPRSS2:ERG fusion and PTEN deletions (P<0.0001 each). High LIG4 expression was also tightly related to early biochemical recurrence when all tumors (P<0.0001) or the subsets of ERG-negative (P=0.0004) or ERG-positive prostate cancers (P=0.006) were analyzed. Multivariate analysis including parameters that are available before surgery demonstrated independent association with biochemical recurrence for advanced Gleason grade on biopsy, high preoperative PSA level, high clinical stage (P<0.0001 each) and for LIG4 immunostaining (P=0.03). Our study identifies LIG4 as a predictor of an increased risk for early PSA recurrence in prostate cancer. Moreover, the strong association with TMPRSS2:ERG fusion and PTEN deletions suggest important interactions between these pathways in prostate cancers.

Cadmium delays non-homologous end joining (NHEJ) repair via inhibition of DNA-PKcs phosphorylation and downregulation of XRCC4 and Ligase IV.

Although studies have shown that cadmium (Cd) interfered with DNA damage repair (DDR), whether Cd could affect non-homologous end joining (NHEJ) repair remains elusive. To further understand the effect of Cd on DDR, we used X-ray irradiation of Hela cells as an in vitro model system, along with γH2AX and 53BP1 as markers for DNA damage. Results showed that X-ray significantly increased γH2AX and 53BP1 foci in Hela cells (p < 0.01), all of which are characteristic of accrued DNA damage. The number of foci declined rapidly over time (1-8h postirradiation), indicating an initiation of NHEJ process. However, the disappearance of γH2AX and 53BP1 foci was remarkably slowed by Cd pretreatment (p < 0.01), suggesting that Cd reduced the efficiency of NHEJ. To further elucidate the mechanisms of Cd toxicity, several markers of NHEJ pathway including Ku70, DNA-PKcs, XRCC4 and Ligase IV were examined. Our data showed that Cd altered the phosphorylation of DNA-PKcs, and reduced the expression of both XRCC4 and Ligase IV in irradiated cells. These observations are indicative of the impairment of NHEJ-dependent DNA repair pathways. In addition, zinc (Zn) mitigated the effects of Cd on NHEJ, suggesting that the Cd-induced NHEJ alteration may partly result from the displacement of Zn or from an interference with the normal function of Zn-containing proteins by Cd. Our findings provide a new insight into the toxicity of Cd on NHEJ repair and its underlying mechanisms in human cells.

XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation.

The classic nonhomologous end-joining (c-NHEJ) pathway is largely responsible for repairing double-strand breaks (DSBs) in mammalian cells. XLF stimulates the XRCC4/DNA ligase IV complex by an unknown mechanism. XLF interacts with XRCC4 to form filaments of alternating XRCC4 and XLF dimers that bridge DNA ends in vitro, providing a mechanism by which XLF might stimulate ligation. Here, we characterize two XLF mutants that do not interact with XRCC4 and cannot form filaments or bridge DNA in vitro. One mutant is fully sufficient in stimulating ligation by XRCC4/Lig4 in vitro; the other is not. This separation-of-function mutant (which must function as an XLF homodimer) fully complements the c-NHEJ deficits of some XLF-deficient cell strains but not others, suggesting a variable requirement for XRCC4/XLF interaction in living cells. To determine whether the lack of XRCC4/XLF interaction (and potential bridging) can be compensated for by other factors, candidate repair factors were disrupted in XLF- or XRCC4-deficient cells. The loss of either ATM or the newly described XRCC4/XLF-like factor, PAXX, accentuates the requirement for XLF. However, in the case of ATM/XLF loss (but not PAXX/XLF loss), this reflects a greater requirement for XRCC4/XLF interaction.

p53 isoform Δ113p53/Δ133p53 promotes DNA double-strand break repair to protect cell from death and senescence in response to DNA damage.

The inhibitory role of p53 in DNA double-strand break (DSB) repair seems contradictory to its tumor-suppressing property. The p53 isoform Δ113p53/Δ133p53 is a p53 target gene that antagonizes p53 apoptotic activity. However, information on its functions in DNA damage repair is lacking. Here we report that Δ113p53 expression is strongly induced by γ-irradiation, but not by UV-irradiation or heat shock treatment. Strikingly, Δ113p53 promotes DNA DSB repair pathways, including homologous recombination, non-homologous end joining and single-strand annealing. To study the biological significance of Δ113p53 in promoting DNA DSB repair, we generated a zebrafish Δ113p53(M/M) mutant via the transcription activator-like effector nuclease technique and found that the mutant is more sensitive to γ-irradiation. The human ortholog, Δ133p53, is also only induced by γ-irradiation and functions to promote DNA DSB repair. Δ133p53-knockdown cells were arrested at the G2 phase at the later stage in response to γ-irradiation due to a high level of unrepaired DNA DSBs, which finally led to cell senescence. Furthermore, Δ113p53/Δ133p53 promotes DNA DSB repair via upregulating the transcription of repair genes rad51, lig4 and rad52 by binding to a novel type of p53-responsive element in their promoters. Our results demonstrate that Δ113p53/Δ133p53 is an evolutionally conserved pro-survival factor for DNA damage stress by preventing apoptosis and promoting DNA DSB repair to inhibit cell senescence. Our data also suggest that the induction of Δ133p53 expression in normal cells or tissues provides an important tolerance marker for cancer patients to radiotherapy.

Expression, crystallization and preliminary crystallographic data analysis of PigI, a putative L-prolyl-AMP ligase from the prodigiosin synthetic pathway in Serratia.

Prodigiosin, a member of the prodiginines, is a tripyrrole red pigment synthesized by Serratia and some other microbes. A bifurcated biosynthesis pathway of prodigiosin has been proposed in Serratia in which MBC (4-methoxy-2,2'-bipyrrole-5-carbaldehyde) and MAP (2-methyl-3-N-amyl-pyrrole) are synthesized separately and then condensed by PigC to form prodigiosin. The first step for the synthesis of MBC is the activation of L-proline by PigI, but its catalytic mechanism has remained elusive. To elucidate its mechanism, recombinant PigI was purified and crystallized. Crystals obtained by the sitting-drop method belonged to space group P1 and diffracted to 2.0 Å resolution, with unit-cell parameters a = 51.2, b = 62.8, c = 91.3 Å, α = 105.1, ß = 90.1, γ = 92.2°. Matthews coefficient analysis suggested two molecules in the asymmetric unit, with a VM of 2.6 Å(3) Da(-1) and a solvent content of 52.69%.

Nucleophosmin modulates stability, activity, and nucleolar accumulation of base excision repair proteins.

Nucleophosmin (NPM1) is a multifunctional protein that controls cell growth and genome stability via a mechanism that involves nucleolar-cytoplasmic shuttling. It is clear that NPM1 also contributes to the DNA damage response, yet its exact function is poorly understood. We recently linked NPM1 expression to the functional activation of the major abasic endonuclease in mammalian base excision repair (BER), apurinic/apyrimidinic endonuclease 1 (APE1). Here we unveil a novel role for NPM1 as a modulator of the whole BER pathway by 1) controlling BER protein levels, 2) regulating total BER capacity, and 3) modulating the nucleolar localization of several BER enzymes. We find that cell treatment with the genotoxin cisplatin leads to concurrent relocalization of NPM1 and BER components from nucleoli to the nucleoplasm, and cellular experiments targeting APE1 suggest a role for the redistribution of nucleolar BER factors in determining cisplatin toxicity. Finally, based on the use of APE1 as a representative protein of the BER pathway, our data suggest a function for BER proteins in the regulation of ribogenesis.

Multiple facets of the DNA damage response contribute to the radioresistance of mouse mesenchymal stromal cell lines.

The regeneration of the hematopoietic system following total body irradiation is supported by host-derived mesenchymal stromal cells (MSCs) within the bone marrow. The mechanisms used by MSCs to survive radiation doses that are lethal to the hematopoietic system are poorly understood. The DNA damage response (DDR) represents a cohort of signaling pathways that enable cells to execute biological responses to genotoxic stress. Here, we examine the role of the DDR in mediating the resistance of MSCs to ionizing radiation (IR) treatment using two authentic clonal mouse MSC lines, MS5 and ST2, and primary bulk mouse MSCs. We show that multiple DDR mechanisms contribute to the radio-resistance of MSCs: robust DDR activation via rapid γ-H2AX formation, activation of effective S and G(2)/M DNA damage checkpoints, and efficient repair of IR-induced DNA double-strand breaks. We show that MSCs are intrinsically programmed to maximize survival following IR treatment by expressing high levels of key DDR proteins including ATM, Chk2, and DNA Ligase IV; high levels of the anti-apoptotic, Bcl-2 and Bcl-(XL); and low levels of the pro-apoptotic, Bim and Puma. As a result, we demonstrate that irradiated mouse MSCs withstand IR-induced genotoxic stress, continue to proliferate, and retain their capacity to differentiate long-term along mesenchymal-derived lineages. We have shown, for the first time, that the DDR plays key roles in mediating the radioresistance of mouse MSCs which may have important implications for the study and application of MSCs in allogeneic bone marrow transplantation, graft-versus-host disease, and cancer treatment.

Expression, purification and biochemical characterization of Methanocaldococcus jannaschii DNA ligase.

We describe the biochemical characterization of Methanocaldococcus jannaschii (M. jannaschii) DNA ligase and its potential application in single nucleotide polymorphism (SNP) genotyping. The recombinant M. jannaschii DNA ligase is an ATP-dependent ligase. The ligase activity was dependent on metal ions of Mg(2+) and Mn(2+). The optimal concentrations of ATP cofactor and Mg(2+) ion were 0.01-2 and 10 mM, respectively. The optimal pH value for DNA ligation was 8.5. High concentrations of NaCl inhibited DNA ligation. The effects of mismatches on joining short oligonucleotides by M. jannaschii DNA ligase were fully characterized. The mismatches at the first position 5' to the nick inhibited ligation more than those at the first position 3' to the nick. The mismatches at other positions 5' to the nick (3rd to 7th sites) exhibited less inhibition on ligation. However, the introduction of a C/C mismatch at the third position 5' to the nick could completely inhibit the ligation of the terminal-mismatched nick of an oligonucleotide duplex by M. jannaschii DNA ligase. Therefore, introducing an additional mismatch at the third position 5' to the SNP site is a more effective approach in genotyping by M. jannaschii DNA ligase.

Lithium chloride protects retinal neurocytes from nutrient deprivation by promoting DNA non-homologous end-joining.

Lithium chloride is a therapeutic agent for treatment of bipolar affective disorders. Increasing numbers of studies have indicated that lithium has neuroprotective effects. However, the molecular mechanisms underlying the actions of lithium have not been fully elucidated. This study aimed to investigate whether lithium chloride produces neuroprotective function by improving DNA repair pathway in retinal neurocyte. In vitro, the primary cultured retinal neurocytes (85.7% are MAP-2 positive cells) were treated with lithium chloride, then cultured with serum-free media to simulate the nutrient deprived state resulting from ischemic insult. The neurite outgrowth of the cultured cells increased significantly in a dose-dependent manner when exposed to different levels of lithium chloride. Genomic DNA electrophoresis demonstrated greater DNA integrity of retinal neurocytes when treated with lithium chloride as compared to the control. Moreover, mRNA and protein levels of Ligase IV (involved in DNA non-homologous end-joining (NHEJ) pathway) in retinal neurocytes increased with lithium chloride. The end joining activity assay was performed to determine the role of lithium on NHEJ in the presence of extract from retinal neurocytes. The rejoining levels in retinal neurocytes treated with lithium were significantly increased as compared to the control. Furthermore, XRCC4, the Ligase IV partner, and the transcriptional factor, CREB and CTCF, were up-regulated in retinal cells after treating with 1.0mM lithium chloride. Therefore, our data suggest that lithium chloride protects the retinal neural cells from nutrient deprivation in vitro, which may be similar to the mechanism of cell death in glaucoma. The improvement in DNA repair pathway involving in Ligase IV might have an important role in lithium neuroprotection. This study provides new insights into the neural protective mechanisms of lithium chloride.

Choice of a start codon in a single transcript determines DNA ligase 1 isoform production and intracellular targeting in Arabidopsis thaliana.

DNA ligase 1 (AtLIG1) is the only essential DNA ligase activity in Arabidopsis and is implicated in the important processes of DNA replication, repair and recombination and in transgene insertion during Agrobacterium-mediated plant transformations. The mitochondrial and nuclear forms of DNA ligase 1 in Arabidopsis are translated from a single mRNA species through the control of translation initiation from either the first (M1) or second (M2) in-frame AUG codons respectively. Translation from a third in-frame AUG codon (M3) occurs on transcripts in which M1 and M2 are mutagenized to stop codons. Wild-type AtLIG1-GFP constructs (where GFP stands for green fluorescent protein) can be targeted in planta to both the nucleus and mitochondria. AtLIG1-GFP translation from M1 specifically targets the fusion protein only to mitochondria in planta, whereas translation from M2 or M3 targets the fusion protein only to the nucleus. Interestingly, the AtLIG1-GFP fusion protein in which translation is initiated from M1 contains both an N-terminal mtPS (mitochondrial targeting presequence) and a nuclear localization signal; nonetheless, this protein is only targeted to the mitochondria. This result raises intriguing questions on the translational control mechanisms that regulate how the protein products of a single transcript are targeted to more than one cellular compartment.

Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay.

DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the K(m) values for NAD+ (2.75+/-0.1 microM) and the acridinium-ester-labelled DNA substrate (2.5+/-0.2 nM). A study of the pH-dependencies of kcat, K(m) and kcat/K(m) has revealed values of kinetically influential ionizations within the enzyme-substrate complexes (kcat) and free enzyme (kcat/K(m)). In each case, the curves were shown to be composed of one kinetically influential ionization, for k(cat), pK(a)=6.6+/-0.1 and kcat/K(m), pK(a)=7.1+/-0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30+/-0.86 microM for doxorubicin and 1.40+/-0.07 microM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 microl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.

Possible function for virus encoded K+ channel Kcv in the replication of chlorella virus PBCV-1.

The K+ channel Kcv is encoded by the chlorella virus PBCV-1. There is evidence that this channel plays an essential role in the replication of the virus, because both PBCV-1 plaque formation and Kcv channel activity in Xenopus oocytes have similar sensitivities to inhibitors. Here we report circumstantial evidence that the Kcv channel is important during virus infection. Recordings of membrane voltage in the host cells Chlorella NC64A reveal a membrane depolarization within the first few minutes of infection. This depolarization displays the same sensitivity to cations as Kcv conductance; depolarization also requires the intact membrane of the virion. Together these data are consistent with the idea that the virus carries functional K+ channels in the virion and inserts them into the host cell plasma membrane during infection.

Coprinus cinereus DNA ligase I during meiotic development.

DNA ligase I is thought to be essential for DNA replication, repair and recombination, at least in the mitotic cell cycle, but whether this is also the case during the meiotic cell cycle is still obscure. To investigate the role of DNA ligase I during the meiotic cell cycle, we cloned the Coprinus cinereus DNA ligase I cDNA (CcLIG1). Northern blotting analysis indicated that CcLIG1 is expressed not only in the premeiotic S-phase but also during the meiotic cell cycle itself. Especially, intense signals were observed in the leptotene and zygotene stages. Western blotting analysis indicated that CcLIG1 is expressed through the meiotic cell cycle and immunofluorescence also showed CcLIG1 protein staining in meiotic cells. Interestingly, the patterns was similar to that for the C. cinereus proliferating cell nuclear antigen gene (CcPCNA) and immunoprecipitation analysis suggested that CcPCNA binds to CcLIG1 in crude extracts of meiotic prophase I tissues. Based on these observations, relationships and roles during the meiotic cell cycle are discussed.

Microsatellite instability and suppressed DNA repair enzyme expression in rheumatoid arthritis.

Reactive oxygen and nitrogen are produced by rheumatoid arthritis (RA) synovial tissue and can potentially induce mutations in key genes. Normally, this process is prevented by a DNA mismatch repair (MMR) system that maintains sequence fidelity during DNA replication. Key members of the MMR system include MutSalpha (hMSH2 and hMSH6) and MutSbeta (hMSH2 and hMSH3). To provide evidence of DNA damage in inflamed synovium, we analyzed synovial tissues for microsatellite instability (MSI). MSI was examined by PCR on genomic DNA of paired synovial tissue and peripheral blood cells of RA patients using specific primer sequences for five key microsatellites. Surprisingly, abundant MSI was observed in RA synovium compared with osteoarthritis tissue. Western blot analysis for the expression of MMR proteins demonstrated decreased hMSH6 and increased hMSH3 in RA synovium. To evaluate potential mechanisms of MMR regulation in arthritis, fibroblast-like synoviocytes (FLS) were isolated from synovial tissues and incubated with the NO donor S-nitroso-N-acetylpenicillamine. Western blot analysis demonstrated constitutive expression of hMSH2, 3, and 6 in RA and osteoarthritis FLS. When FLS were cultured with S-nitroso-N-acetylpenicillamine, the pattern of MMR expression in RA synovium was reproduced (high hMSH3, low hMSH6). Therefore, oxidative stress can relax the DNA MMR system in RA by suppressing hMSH6. Decreased hMSH6 can subsequently interfere with repair of single base mutations, which is the type observed in RA. We propose that oxidative stress not only creates DNA adducts that are potentially mutagenic, but also suppresses the mechanisms that limit the DNA damage.

Proteolysis of a nucleotide excision repair protein by the 26 S proteasome.

The 26 S proteasome degrades a broad spectrum of proteins and interacts with several nucleotide excision repair (NER) proteins, including the complex of Rad4 and Rad23 that binds preferentially to UV-damaged DNA. The rate of NER is increased in yeast strains with mutations in genes encoding subunits of the 26 S proteasome, indicating that it could negatively regulate a repair process. The specific function of the 26 S proteasome in DNA repair is unclear. It might degrade DNA repair proteins after repair is completed or act as a molecular chaperone to promote the assembly or disassembly of the repair complex. In this study, we show that Rad4 is ubiquitylated and that Rad23 can control this process. We also find that ubiquitylated Rad4 is degraded by the 26 S proteasome. However, the interaction of Rad23 with Rad4 is not only to control degradation of Rad4, but also to assist in assembling the NER incision complex at UV-induced cyclobutane pyrimidine dimers. We speculate that, following the completion of DNA repair, specific repair proteins might be degraded by the proteasome to regulate repair.

Induction of DNA ligase I by 1-beta-D-arabinosylcytosine and aphidicolin in MiaPaCa human pancreatic cancer cells.

Exposure of MiaPaCa cells to 1-beta-D-arabinosylcytosine (ara-C) resulted in an increase in DNA ligase levels up to threefold compared to that in the untreated control cells, despite significant growth inhibition. Increased levels of DNA ligase I protein appear to correlate with the appearance of increased mRNA levels. The [(3)H]thymidine incorporation experiment and the biochemical assay of total polymerase activity revealed that an increase in DNA ligase I levels after treatment with ara-C was not accompanied by an increase of DNA synthesis or an increased presence of DNA polymerase activity inside cells. When cells resumed DNA synthesis after drug treatment, DNA ligase I levels began to drop, indicating that increased DNA ligase I is not required for DNA synthesis. An increase in DNA ligase I was also observed in cells treated with aphidicolin, another inhibitor of DNA synthesis that inhibits DNA polymerases without incorporating itself into DNA, indicating that an increase in DNA ligase I levels could be caused by the arrest of DNA replication by these agents. Interestingly, caffeine, which is a well-known inhibitor of DNA damage checkpoint kinases, abrogated the increase in DNA ligase I in MiaPaCa cells treated with ara-C and aphidicolin, suggesting that caffeine-sensitive kinases might be important mediators in the pathway leading to the increase in DNA ligase I levels in response to anticancer drugs, including ara-C and aphidicolin. We propose that ara-C and aphidicolin induce damage to the DNA strand by arresting DNA replication forks and subsequently increase DNA ligase I levels to facilitate repair of DNA damage.

Inter-individual variation in urothelial DNA repair gene expression in vitro.

DNA repair efficiency may play a significant role in individual susceptibility to bladder cancer, the third most common cancer in Europe. Bladder cancer arises from the urothelial cell layer which lines the urinary tract. As DNA repair gene expression levels should reflect DNA repair capacity, we investigated the expression of genes from the base excision, nucleotide excision and mismatch repair pathways in normal human urothelial (NHU) cells in vitro. RNA was extracted from six independent NHU cell lines and expression of 26 DNA repair genes was determined by ribonuclease protection assay. The results show that all the genes analysed were detected in NHU cells in vitro with a similar expression pattern in most cell lines. However, there was some variation between cell lines, with one expressing base excision repair genes very strongly, but another having weak expression of mismatch repair genes. These results suggest that DNA repair genes are constitutively expressed by NHU cells and that there is some inter-individual variation. Prospective studies are required to determine whether these differences in gene expression may play a role in susceptibility to bladder cancer.

Counteracting spontaneous transformation via overexpression of rate-limiting DNA base excision repair enzymes.

DNA damage of endogenous origin may significantly contribute to human cancer. A major pathway involved in DNA repair of endogenous damage is DNA base excision repair (BER). BER is rather efficient in human cells but a certain amount of endogenous damage inevitably escapes mending and likely contributes to human carcinogenesis. Apart from some glycosylases that are particularly sluggish (e.g. 8-oxoG DNA glycosylase), recent work suggests that the general rate-limiting steps of BER may be trimming of 2-deoxyribose 5-phosphate in case the process is started by a monofunctional glycosylase or trimming of a 3'-blocking fragment, in case BER is started by a bifunctional glycosylase or in the case of single-strand breaks produced by free radical attack. Overexpression of the 5'-deoxyribophosphodiesterase (dRPase) domain of DNA polymerase beta, on the one hand, and of yeast APN1 protein, containing an efficient 3' repair activity, on the other, may lead to improved BER in mammals. The recently characterized S3 protein of Drosophila, containing both dRPase and 3'-trimming activities, could also be considered for overexpression studies. The possible protecting role of enhanced BER could be investigated in cultured rodent embryonic fibroblasts undergoing spontaneous transformation, a most interesting system that merits rediscovery.

NAD+-dependent DNA ligase encoded by a eukaryotic virus.

We report the production, purification, and characterization of an NAD(+)-dependent DNA ligase encoded by the Amsacta moorei entomopoxvirus (AmEPV), the first example of an NAD(+) ligase from a source other than eubacteria. AmEPV ligase lacks the zinc-binding tetracysteine domain and the BRCT domain that are present in all eubacterial NAD(+) ligases. Nonetheless, the monomeric 532-amino acid AmEPV ligase catalyzed strand joining on a singly nicked DNA in the presence of a divalent cation and NAD(+). Neither ATP, dATP, nor any other nucleoside triphosphate could substitute for NAD(+). Structure probing by limited proteolysis showed that AmEPV ligase is punctuated by a surface-accessible loop between the nucleotidyltransferase domain, which is common to all ligases, and the N-terminal domain Ia, which is unique to the NAD(+) ligases. Deletion of domain Ia of AmEPV ligase abolished the sealing of 3'-OH/5'-PO(4) nicks and the reaction with NAD(+) to form ligase-adenylate, but had no effect on phosphodiester formation at a pre-adenylated nick. Alanine substitutions at residues within domain Ia either reduced (Tyr(39), Tyr(40), Asp(48), and Asp(52)) or abolished (Tyr(51)) sealing of a 5'-PO(4) nick and adenylyl transfer from NAD(+) without affecting ligation of DNA-adenylate. We conclude that: (i) NAD(+)-dependent ligases exist in the eukaryotic domain of the phylogenetic tree; and (ii) ligase structural domain Ia is a determinant of cofactor specificity and is likely to interact directly with the nicotinamide mononucleotide moiety of NAD(+).

Efficient recognition of substrates and substrate analogs by the adenine glycosylase MutY requires the C-terminal domain.

The Escherichia coli DNA repair enzyme MutY plays an important role in the prevention of DNA mutations by removing misincorporated adenine residues from 7, 8-dihydro-8-oxo-2'-deoxyguanosine:2'-deoxyadenosine (OG:A) mispairs. The N-terminal domain of MutY (Stop 225, Met1-Lys225) has a sequence and structure that is characteristic of a superfamily of base excision repair glycosylases; however, MutY and its homologs contain a unique C-terminal domain. Previous studies have shown that the C-terminal domain confers specificity for OG:A substrates over G:A substrates and exhibits homology to the d(OG)TPase MutT, suggesting a role in OG recognition. In order to provide additional information on the importance of the C-terminal domain in damage recognition, we have investigated the kinetic properties of a form lacking this domain (Stop 225) under multiple- and single-turnover conditions. In addition, the interaction of Stop 225 with a series of non-cleavable substrate and product analogs was evaluated using gel retardation assays and footprinting experiments. Under multiple-turnover conditions Stop 225 exhibits biphasic kinetic behavior with both OG:A and G:A substrates, likely due to rate-limiting DNA product release. However, the rate of turnover of Stop 225 was increased 2-fold with OG:A substrates compared to the wild-type enzyme. In contrast, the intrinsic rate for adenine removal by Stop 225 from both G:A and OG:A substrates is significantly reduced (10- to 25-fold) compared to the wild-type. The affinity of Stop 225 for substrate analogs was dramatically reduced, as was the ability to discriminate between substrate analogs paired with OG over G. Interestingly, similar hydroxyl radical and DMS footprinting patterns are observed for Stop 225 and wild-type MutY bound to DNA duplexes containing OG opposite an abasic site mimic or a non-hydrogen bonding A analog, suggesting that similar regions of the DNA are contacted by both enzyme forms. Importantly, Stop 225 has a reduced ability to prevent DNA mutations in vivo. This implies that the reduced adenine glycosylase activity translates to a reduced capacity of Stop 225 to prevent DNA mutations in vivo.