Initiate Excision Repair Research Articles

Large-Scale Photolithographic Synthesis of Chimeric DNA/RNA Hairpin Microarrays To Explore Sequence Specificity Landscapes of RNase HII Cleavage.

Ribonuclease HII (RNase HII) is an essential endoribonuclease that binds to double-stranded DNA with RNA nucleotide incorporations and cleaves 5′ of the ribonucleotide at RNA–DNA junctions. Thought to be present in all domains of life, RNase HII protects genomic integrity by initiating excision repair pathways that protect the encoded information from rapid degradation. There is sparse evidence that the enzyme cleaves some substrates better than others, but a large-scale study is missing. Such large-scale studies can be carried out on microarrays, and we employ chemical photolithography to synthesize very large combinatorial libraries of fluorescently labeled DNA/RNA chimeric sequences that self-anneal to form hairpin structures that are substrates for Escherichia coli RNase HII. The relative activity is determined by the loss of fluorescence upon cleavage. Each substrate includes a double-stranded 5 bp variable region with one to five consecutive ribonucleotide substitutions. We also examined the effect of all possible single and double mismatches, for a total of >9500 unique structures. Differences in cleavage efficiency indicate some level of substrate preference, and we identified the 5′-dC/rC-rA-dX-3′ motif in well-cleaved substrates. The results significantly extend known patterns of RNase HII sequence specificity and serve as a template using large-scale photolithographic synthesis to comprehensively map landscapes of substrate specificity of nucleic acid-processing enzymes.

Analysis of SHPRH functions in DNA repair and immunoglobulin diversification.

During replication, bypass of DNA lesions is orchestrated by the Rad6 pathway. Monoubiquitination of proliferating cell nuclear antigen (PCNA) by Rad6/Rad18 leads to recruitment of translesion polymerases for direct and potentially mutagenic damage bypass. An error-free bypass pathway may be initiated via K63-linked PCNA polyubiquitination by Ubc13/Mms2 and the E3 ligase Rad5 in yeast, or HLTF/SHPRH in vertebrates. For the latter two enzymes, redundancy with a third E3 ligase and alternative functions have been reported. We have previously shown that the Rad6 pathway is involved in somatic hypermutation of immunoglobulin genes in B lymphocytes. Here, we have used knockout strategies targeting expression of the entire SHPRH protein or functionally significant domains in chicken DT40 cells that do not harbor a HLTF ortholog. We show that SHPRH is apparently redundant with another E3 ligase during DNA damage-induced PCNA modification. SHPRH plays no substantial role in cellular resistance to drugs initiating excision repair and the Rad6 pathway, but is important in survival of topoisomerase II inhibitor treatment. Removal of only the C-terminal RING domain does not interfere with this SHPRH function. SHPRH inactivation does not substantially impact on the overall efficacy of Ig diversification. Redundancy of E3 ligases in the Rad6 pathway may be linked to its different functions in genome maintenance and genetic plasticity.

Alternative Excision Repair Pathways

Alternative excision repair (AER) is a category of excision repair initiated by a single nick, made by an endonuclease, near the site of DNA damage, and followed by excision of the damaged DNA, repair synthesis, and ligation. The ultraviolet (UV) damage endonuclease in fungi and bacteria introduces a nick immediately 5' to various types of UV damage and initiates its excision repair that is independent of nucleotide excision repair (NER). Endo IV-type apurinic/apyrimidinic (AP) endonucleases from Escherichia coli and yeast and human Exo III-type AP endonuclease APEX1 introduce a nick directly and immediately 5' to various types of oxidative base damage besides the AP site, initiating excision repair. Another endonuclease, endonuclease V from bacteria to humans, binds deaminated bases and cleaves the phosphodiester bond located 1 nucleotide 3' of the base, leading to excision repair. A single-strand break in DNA is one of the most frequent types of DNA damage within cells and is repaired efficiently. AER makes use of such repair capability of single-strand breaks, removes DNA damage, and has an important role in complementing BER and NER.

Diagnostic correlation between the expression of the DNA repair enzymeN-methylpurine DNA glycosylase and esophageal adenocarcinoma onset: a retrospective pilot study

EAC in its early stages, when it can potentially be cured, is rarely symptomatic and is associated with high mortality rates because in part of late-stage diagnosis. Given that DNA repair is an important contributory factor of early-stage malignancy, our study focused on the expression of the base excision repair enzyme N-methylpurine DNA glycosylase (MPG) in EAC disease onset. MPG messenger RNA (mRNA) expression levels were determined using quantitative reverse transcriptase polymerase chain reaction from a maximum of 72 patient samples. Immunohistochemistry was further utilized for the detection of MPG protein, and semiquantitative analysis performed using an H-score approach was carried out on a total of 130 archival tissue samples of different esophageal pathologies. Nuclear localized MPG protein was detected in all nonmalignant tissues derived from the enterohepatic system, with H-score values of 3.9-5.5 ± 0.4-1.0. In cancerous tissues derived from the enterohepatic system, a 9.5-fold increase in the level of MPG mRNA expression was specifically observed in the malignant regions located within the esophagus region. Further analysis revealed a 9- and 14-fold increase in MPG mRNA expression in EAC tumor, node, metastasis stages II and III, respectively, suggesting MPG expression to correlate with EAC disease progression. Immunohistochemistry analysis further showed a sevenfold significant increase in MPG protein expression in EAC tissues. Intriguingly, there was a fivefold significant decrease in nuclear localized MPG protein expression in tissues derived from Barrett's esophagus and low-grade dysplasia. Such findings highlight a complex regulatory pattern governing DNA glycosylase base excision repair initiation, as normal tissue undergoes Barrett's metaplasia and later dedifferentiates to EAC. Indeed, disease-stage-specific alterations in the expression of MPG may highlight a potential role for MPG in determining EAC onset and thus potentially be of clinical relevance for early disease detection and increased patient survival.

Production of clastogenic DNA precursors by the nucleotide metabolism inEscherichia coli

RdgB is a bacterial dNTPase with a strong in vitro preference for non-canonical DNA precursors dHapTP, dXTP and dITP that contain deaminated or aminogroup-modified purines. Utilization of these nucleotides by replisomes in rdgB mutants of Escherichia coli produces modified DNA, on which EndoV nicking near the base analogues initiates excision repair. Some EndoV-initiated excision events cause chromosomal fragmentation, which becomes inhibitory if recombinational repair is also inactivated (the rdgB recA co-inhibition). To reveal the sources and the identities of the non-canonical DNA precursors, intercepted by RdgB in E. coli, we characterized 17 suppressors of the rdgB recA co-inhibition. Ten suppressors affect genes of the RNA/DNA precursor metabolism, identifying the source of non-canonical DNA precursors. Comparing chromosomal fragmentation with the density of EndoV-recognized DNA modifications distinguishes three mechanisms of suppression: (i) reduction of the non-canonical dNTP production, (ii) inhibition of the base analogue excision from DNA and (iii) enhancement of the cell tolerance to chromosomal fragmentation. The suppressor analysis suggests IMP as the key intermediate in the synthesis of the clastogenic DNA precursor, most likely dITP.

Expression, purification and characterization of codon-optimized human N-methylpurine-DNA glycosylase from Escherichia coli

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, initiates excision repair of several N-alkylpurine adducts, deaminated and lipid peroxidation-induced purine adducts. MPG from human and mouse has previously been cloned and expressed. However, due to the poor expression level in Escherichia coli (E. coli) and multi-step purification process of full-length MPG, most successful attempts have been limited by extremely poor yield and stability. Here, we have optimized the codons within the first five residues of human MPG (hMPG) to the best used codons for E. coli and expressed full-length hMPG in large amounts. This high expression level in conjunction with a strikingly high isoelectric point (9.65) of hMPG, in fact, helped purify the enzyme in a single step. A previously well-characterized monoclonal antibody having an epitope in the N-terminal tail could detect this codon-optimized hMPG protein. Surface plasmon resonance studies showed an equilibrium binding constant (KD) of 0.25nM. Steady-state enzyme kinetics showed an apparent Km of 5.3nM and kcat of 0.2min−1 of MPG for the hypoxanthine (Hx) cleavage reaction. Moreover, hMPG had an optimal activity at pH 7.5 and 100mM KCl. Unlike the previous reports by others, this newly purified full-length hMPG is appreciably stable at high temperature, such as 50°C. Thus, this study indicates that this improved expression and purification system will facilitate large scale production and purification of a stable human MPG protein for further biochemical, biophysical and structure–function analysis.

Discrimination of lesion removal of N-methylpurine-DNA glycosylase revealed by a potent neutralizing monoclonal antibody

N-Methylpurine-DNA glycosylase (MPG), a ubiquitous DNA repair enzyme, initiates excision repair of several N-alkylpurine adducts, induced by alkylating chemotherapeutics, and deaminated and lipid peroxidation-induced purine adducts. We have generated monoclonal antibodies (moAbs) against human MPG. Twelve independent hybridoma clones were characterized, which, except 520-16A, are identical based on epitope exclusion assay. Four moAbs, including 520-2A, 520-3A, 520-16A, and 520-26A, have high affinity ( K D ∼ 0.3–1.6 nM), and their subtypes were IgG 2a, IgG 1, IgG 2a, and IgG 2b, respectively. moAb 520-3A recognizes the sequence 52AQAPCPRERCLGPP 66T, an epitope exclusively present in the N-terminal extension of human MPG. We found that moAb 520-3A significantly inhibited MPG's enzymatic activity towards different substrates, such as hypoxanthine, 1, N 6ethenoadenine and methylated bases, which represent different classes of DNA damage, however, with different efficiencies. Real-time binding experiments using surface plasmon resonance (SPR) spectroscopy showed that the pronounced inhibition of activity was not in the substrate-binding step. Single turnover kinetics (STO) revealed that the inhibition was at the catalytic step. Since we found that this antibody has an epitope in the N-terminal tail, the latter appears to have an important role in substrate discrimination, however, with a differential effect on different substrates.

Ubiquitylation-independent degradation of Xeroderma pigmentosum group C protein is required for efficient nucleotide excision repair

The Xeroderma Pigmentosum group C (XPC) protein is indispensable to global genomic repair (GGR), a subpathway of nucleotide excision repair (NER), and plays an important role in the initial damage recognition. XPC can be modified by both ubiquitin and SUMO in response to UV irradiation of cells. Here, we show that XPC undergoes degradation upon UV irradiation, and this is independent of protein ubiquitylation. The subunits of DDB-Cul4A E3 ligase differentially regulate UV-induced XPC degradation, e.g DDB2 is required and promotes, whereas DDB1 and Cul4A protect the protein degradation. Mutation of XPC K655 to alanine abolishes both UV-induced XPC modification and degradation. XPC degradation is necessary for recruiting XPG and efficient NER. The overall results provide crucial insights regarding the fate and role of XPC protein in the initiation of excision repair.

Human DNA polymerase beta initiates DNA synthesis during long-patch repair of reduced AP sites in DNA.

Simple base damages are repaired through a short-patch base excision pathway where a single damaged nucleotide is removed and replaced. DNA polymerase beta (Pol beta) is responsible for the repair synthesis in this pathway and also removes a 5'-sugar phosphate residue by catalyzing a beta-elimination reaction. How ever, some DNA lesions that render deoxyribose resistant to beta-elimination are removed through a long-patch repair pathway that involves strand displacement synthesis and removal of the generated flap by specific endonuclease. Three human DNA polymerases (Pol beta, Pol delta and Pol epsilon) have been proposed to play a role in this pathway, however the identity of the polymerase involved and the polymerase selection mechanism are not clear. In repair reactions catalyzed by cell extracts we have used a substrate containing a reduced apurinic/apyrimidinic (AP) site resistant to beta-elimination and inhibitors that selectively affect different DNA polymerases. Using this approach we find that in human cell extracts Pol beta is the major DNA polymerase incorporating the first nucleotide during repair of reduced AP sites, thus initiating long-patch base excision repair synthesis.

Effects of benzo(a)pyrenf and tetrachlorodibenzo(p) dioxin on fetal dolphin kidney cells: Inhibition of proliferation and initiation of DNA damage

Dolphin kidney cells (CDK) were exposed in vitro to benzo(a)pyrene (BaP) in the presence or absence of 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD), a cytochrome P450-inducing agent, and/or α-naphthoflavone (α NF), an inhibitor of cytochrome P450 induction. BaP inhibited mitosis in CDK cells in a dose-dependent manner. TCDD, while inhibiting cell proliferation, did not show a strict dose-dependent mode of action. BaP inhibition of mitosis was decreased by αNF, which also decreased the inhibitory effects of TCDD on CDK proliferation. BaP treatment initiated both 3H-thymidine incorporation and the increased alkali lability of DNA functions of the initiation of excision repair. Cells pre-treated with TCDD and then exposed to BaP exhibited increased BaP-DNA adduct levels and increased DNA excision repair. These data indicate that dolphin cells metabolized BaP in vitro as a function of cytochrome P450-associated activities, that BaP metabolites covalently bound to cellular DNA and initiated excision repair. Inhibition of the cytochrome P450-mediated metabolism of BaP decreased the BaP-associated inhibition of mitosis in dolphin cells.

Enhanced unscheduled DNA synthesis by secondary cultures of lung cells established from calorically restricted aged rats

Unscheduled DNA synthesis (UDS) induced by two exposure levels of ultraviolet light (UV) or two concentrations of methyl methane sulfonate (MMS) was evaluated in secondary cultures of lung fibroblasts established from weanling, 11-month-old and 31-month-old female Fischer 344 rats fed and libitum (AL) or calorically restricted (CR) diets. [ 3H]Thymidine incorporation as a function of UDS was highest for weanling-derived cells treated with either UV or MMS, declining consistently with increased age between cells from weanling, 11-month-old and 31-month-old animals. [ 3H]Thymidine incorporation as a function of UDS in cells from 11-month-old AL vs. CR rats differed only at the highest UV exposure level. In contrast, cells drived from 31-month-old CR rats exhibited UDS levels which were at leastt wice as high at each UV treatment level as UDS levels of cells drived from the same age AL rats. Cells from both old AL and old CR rats were shown to initiate DNA excision repair at about the same rate. Cells from CR rats, however, repaired DNA damage at an accelerated rate and completed excision repair while repair in cells from AL animals was slower and apparently did not proceed to completion. Data from this study indicate that cells from young and old AL and CR animals initiate excision repair, but demonstrate an age-related loss of UV- or MMS-stimulated [ 3H]thymidine incorporation in cells derived from AL animals. Cells derived from CR animals did not exhibit that age-related loss of UDS activity; rather, they showed an enhanced UDS response to DNA damage and appeared to complete ligation as the final step in excision repair. The data suggest that caloric restriction of a cell donor animal not only delays the age-associated decrease in in vitro DNA excision repair capacity in cells from that animal, but may actually enhance repair capacity.

Enhanced pyrimidine dimer removal in repair-proficient murine fibroblasts transforme with the denV gene of bacteriophage T4

The denV gene of bacteriophage T4, which encodes the pyrimidine dimer-specific repair enzyme endonuclease V, was introduced into murine fibroblasts with normal rodent pyrimidine dimer repair capabilities. Endonuclease V recognizes ultraviolet radiation (UVR)-induced pyrimidine dimers and produces single-strand breaks adjacent to the dimers. These nicks may serve as substrates to initiate excision repair fo pyrimidine dimers endogenous enzymes. In the present study, murine fibroblasts stably transfered with denV were able to remove 50–80% of UVR-induced pyramidine dimers, while control cells removed only about 20% of dimers under the same conditions of pyrimidine dimer induction and repair. For both control and denV-transfected cells, repair continued for at least 24 h after exposure. When removal of UVR-induced photoproducts was initiated by endogenous excision repair mechanisms, an average of 38 nucleotides were replaced per dimer removed, as determined by bromouracil photolysis; denV-initiated excision repair, on the other hand, resulted in removal of an average of 6 nucleotides per dimer repaired. The enhanced pyrimidine diner repair capabilities conferred by denV gene expression did not appear to improve post-UVR survival.

Preferential recognition of I·T base-pairs in the initiation of excision-repair by hypoxanthine-DNA glycosylase

Double-stranded synthetic oligonucleotides with a centrally located dIMP residue in a 5'-32P-labeled strand were employed as substrates for hypoxanthine-DNA glycosylase. The enzyme activity was monitored by the generation of a piperidine-sensitive site in the labeled oligonucleotide. The enzyme was purified approximately 5000-fold from calf thymus. The purified enzyme removed efficiently a hypoxanthine base residue from an I.T base pair, but 15-20 times more slowly from an I.C base pair. Similar results were obtained with oligonucleotides in which the deoxyinosine residue was placed in different surrounding nucleotide sequences. The enzyme had no detectable activity on mismatched G.T, A.G or A.C base pairs. The data indicate that hypoxanthine-DNA glycosylase participates in the repair of deaminated adenine residues in DNA.

Construction of a recombinant adenovirus containing the den V gene from bacteriophage T4 which can partially restore the DNA repair deficiency in xeroderma pigmentosum fibroblasts

The denV gene from bacteriophage T4 encodes a pyrimidine dimer-specific endonuclease that has the capacity to initiate excision repair of DNA. Cells from excision repair-deficient xeroderma pigmentosum (XP) patients are able to carry out excision repair initiated by the denV gene product and introduction of the denV gene into XP cells results in the partial restoration of colony-forming ability after irradiation with UV light. In this work we have constructed a helper-independent recombinant human adenovirus, Ad5denV, which contains the denV gene. A 1.9 kb cartridge consisting of the denV gene flanked by the long terminal repeat (LTR) promoter from Rous sarcoma virus (RSV) and the simian virus 40 (SV40) polyadenylation (poly A) splice signals, was inserted into the E3 region of an E3 deletion mutant (Ad5d1E3) of adenovirus type 5. Infection of human fibroblasts and other permissive human cells with Ad5denV resulted in lytic infection and expression of the denV gene was confirmed by primer extension of infected cell RNA. The ability of the denV gene to restore the DNA repair deficiency in XP fibroblasts was examined using host cell reactivation of viral structural antigen formation for UV-irradiated adenovirus. The control virus, Ad5VSV, was also a recombinant which contained the gene for vesicular stomatitis virus glycoprotein G inserted into the E3 region of Ad5d1E3. UV survival of Ad5denV was similar to that of Ad5VSV following infection of two normal fibroblast strains and a Cockayne syndrome fibroblast strain, CS7SE, from complementation group B. In contrast, UV survival of Ad5denV was significantly greater than that for Ad5VSV after infection of three unrelated XP fibroblast strains from complementation groups A, C and E. However, UV survival of Ad5denV in the XP fibroblasts did not reach levels obtained in normal fibroblasts, indicating that restoration of the XP defect was partial.

Differential cell cycle modulation of human DNA glycosylases against oxidized pyrimidines

Cellular DNA is continuously subject to damages by both endogenous and exogenous oxidizing agents. Excision repair of oxidized bases in human cells is initiated by DNA glycosylases which remove them from DNA. 5-Hydroxymethyluracil-DNA glycosylase excises 5-hydroxymethyluracil from DNA. A different enzyme, termed a redoxyendnuclease, has glycosylase activity against many modified DNA pyrimidines. The regulation of these enzymes in proliferating human cells was examined. Both glycosylases were assayed in serum-stimulated WI-38 cells by measurements of direct release of modified free bases from their respective DNA substrates. There was no significant variation of 5-hydroxymethyluracil-DNA glycosylase activity during the cell cycle. However, the glycosylic activity of the redoxyendonuclease was stimulated with DNA synthesis. Thi activity again increased at the beginning of a second cell cycle. Therefore, the glycosylases that initiate excision repair of oxidized DNA are subject to different controls during the cell cycle.

Enhanced repair of pyrimidine dimers in coding and non-coding genomic sequences in CHO cells expressing a prokaryotic DNA repair gene.

We have previously demonstrated that the active dihydrofolate reductase (DHFR) gene is efficiently repaired in Chinese hamster ovary (CHO) cells which remove only a small fraction of u.v.-induced pyrimidine dimers from the overall genome. Preferential DNA repair of essential genes may explain why the u.v. resistance of normal CHO cells is as high as that of fully repair-proficient normal human cells. In this report, we have studied the removal of pyrimidine dimers in a CHO cell line expressing the cloned denV gene from bacteriophage T4 which codes for the pyrimidine dimer specific enzyme T4 endonuclease V (T4 endo V). This cell line was derived from a u.v.-sensitive excision deficient mutant of a CHO wild type line by transformation with the denV gene, and partial restoration of u.v. resistance was achieved. We have examined an important aspect of the u.v. excision repair in these denV+ cells by studying the repair efficiencies in the active DHFR gene and in a non-coding sequence located downstream from it. In the u.v.-sensitive CHO mutant cell line from which the denV+ was derived, we detected no pyrimidine dimer removal from the gene or from the downstream sequence after irradiation of the cells with 20 J/m2 u.v. (254 nm) light. In the wild type CHO cells, approximately 50% of the pyrimidine dimers were removed from a sequence in the DHFR gene within 8 h, whereas none were removed from the downstream sequence in that period. This represents the normal pattern of preferential DNA repair of active genes, which we have described in previous communications. In the denV+ cells, approximately 70% of the pyrimidine dimers were removed from both the DHFR gene and from the downstream sequence; these cells thus repair both coding and non-coding regions of the genome and show no pattern of preferential repair. The endogenous activity that initiates excision repair in normal CHO cells is evidently much more restricted in its accessibility to DNA lesions in chromatin than is the activity in cells containing substantial amounts of the small T4 endo V enzyme.

Human lymphocytes treated with r-7, t-8-dihydroxy- t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene require low-density lipoproteins for DNA excision repair

Human lymphocytes which were non-mitogen-stimulated, and which were depleted of lipoproteins, were found to be deficient in DNA excision repair typically initiated in these cells in response to treatment with a direct-acting polynuclear aromatic hydrocarbon carcinogen. Lymphocytes either depleted of lipoproteins or supplemented with human low-density lipoproteins formed DNA—carcinogen adducts which were not chromatographically distinguishable. The state of lipoprotein depletion did not alter lymphocyte uptake of thymidine from the medium. Lymphocytes which are depleted of lipoproteins, treated with carcinogen, and subsequently supplemented with low-density lipoproteins, regained the ability to engage in DNA excision repair. The data suggest that either low-density lipoprotein(s), or a component(s) of low-density lipoprotein(s), is required by human lymphocytes in order to initiate excision repair of carcinogen-damaged DNA.