DNA Repair in mammalian cells: Mismatched repair: variations on a theme.

Complementary base pairing underlies the genetic template function of the DNA double helix. Therefore, to assure faithful DNA transactions, cells must adhere to a strict application of the Watson-Crick base pairing principle.Yet, mispairing does arise in DNA, most frequently as a result of DNA polymerase errors or base damage. These mismatches need be rectified to avoid mutation. Sometimes, however, mispairing is actively induced to trigger mutagenesis. This happens in activated B-lymphocytes, where the targeted generation and processing of G.U mismatches contributes to somatic hypermutation and antibody diversification. Non-mutagenic mismatches arise in heteroduplex intermediates of homologous recombination, and their processing helps restrict homeologous recombination. Depending on the type of mismatch and the biological context of its occurrence, cells must apply appropriate strategies of repair to properly control mutagenesis. This review will illustrate conceptual and functional challenges of cellular mismatch correction on typical examples of mutagenic base-base mismatches. (Part of a Multi-author Review).

Normal colorectal mucosa exhibits sex- and segment-specific susceptibility to DNA methylation at the hMLH1 and MGMT promoters.

Silencing of gene expression by aberrant cytosine methylation is a prominent feature of human tumors, including colorectal cancers. Epigenetic changes of this type play undisputed roles in cell transformation when they involve genes that safeguard genome stability, and they can also be detected in precancerous lesions and seemingly normal peritumoral tissues. We explored physiological conditions associated with aberrant promoter methylation involving two DNA-repair genes in normal colorectal mucosa. Samples of cecal, transverse colon, sigmoid and rectal mucosa collected from 100 healthy individuals undergoing screening colonoscopy were analysed for hMLH1 and MGMT promoter methylation with a quantitative PCR assay. Positivity in at least one colon segment was common in both sexes, with methylation involving 0.1-18.8% of the alleles (median=0.49%). Samples from males showed no consistent patterns for either promoter, but there were striking age- and colon segment-specific differences in the female subgroup. Here, the prevalence of hMLH1 and MGMT methylation increased significantly with age, particularly in the right colon, where there was also an age-related increase in the percentage of alleles showing hMLH1 methylation. Concomitant methylation of both promoters was also significantly more common in the right colon of women. These findings paralleled immunohistochemical patterns of hMLH1 and MGMT protein loss in an independent series of 231 colorectal cancers and were consistent with current epigenetic profiles of colorectal cancer subsets. They suggest the intriguing possibility that the epigenetic signatures of cancers may have early-stage, normal-tissue counterparts that reflect potentially important aspects of the initial carcinogenetic process.

NEJ1 controls non-homologous end joining in Saccharomyces cerevisiae.

Broken DNA ends are rejoined by non-homologous end-joining (NHEJ) pathways requiring the Ku proteins (Ku70, Ku80), DNA ligase IV and its associated protein Lif1/Xrcc4 (ref. 1). In mammalian meiotic cells, Ku protein levels are much lower than in somatic cells, apparently reducing the capacity of meiotic cells to carry out NHEJ and thereby promoting homologous recombination. In Saccharomyces cerevisiae, NHEJ is also downregulated in meiosis-competent MATa/MAT alpha diploid cells in comparison with diploids or haploids expressing only MATa or MAT alpha. Diploids expressing both MATa and MAT alpha show enhanced mitotic homologous recombination. Here we report that mating-type-dependent regulation of NHEJ in budding yeast is caused in part by transcriptional repression of both LIF1 and the gene NEJ1 (YLR265C)--identified from microarray screening of messenger RNAs. Deleting NEJ1 reduces NHEJ 100-fold in MATa or MAT alpha haploids. Constitutive expression of NEJ1, but not expression of LIF1, restores NHEJ in MATa/MAT alpha cells. Nej1 regulates the subcellular distribution of Lif1. A green fluorescent protein (GFP)-Lif1 fusion protein accumulates in the nucleus in cells expressing NEJ1 but is largely cytoplasmic when NEJ1 is repressed.

Thymine DNA glycosylase.

More than 50% of colon cancer-associated mutations in the p53 tumor suppressor gene are C-->T transitions. The majority of them locate in CpG dinucleotides and are thought to have arisen through spontaneous hydrolytic deamination of 5-methylcytosine. This deamination process gives rise to G.T mispairs that need to be repaired to G.C in order to avoid C-->T mutation. Similarly, deamination of cytosine generates G.U mispairs that also produce C-->T transitions if not repaired. Restoration of both G.T and G.U mismatches was shown to be mediated by a short-patch excision repair pathway, and one principal player implicated in this process may be thymine DNA glycosylase (TDG). Human TDG was discovered as an enzyme that has the potential to specifically remove thymine and uracil bases mispaired with guanine through hydrolysis of their N-glycosidic bond, thereby generating abasic sites in DNA and initiating a base excision repair reaction. The same protein was later found to interact physically and functionally with the retinoid receptors RAR and RXR, and this implicated an unexpected function of TDG in nuclear receptor-mediated transcriptional activation of gene expression. The objective of this chapter is to put together the results of different lines of experimentation that have explored the thymine DNA glycosylase since its discovery and to critically evaluate their implications for possible physiological roles of this enzyme.

Biochemical characterization of uracil processing activities in the hyperthermophilic archaeon Pyrobaculum aerophilum.

Deamination of cytosine to uracil and 5-methylcytosine to thymine represents a major mutagenic threat particularly at high temperatures. In double-stranded DNA, these spontaneous hydrolytic reactions give rise to G.U and G.T mispairs, respectively, that must be restored to G.C pairs prior to the next round of DNA replication; if left unrepaired, 50% of progeny DNA would acquire G.C --> A.T transition mutations. The genome of the hyperthermophilic archaeon Pyrobaculum aerophilum has been recently shown to encode a protein, Pa-MIG, a member of the endonuclease III family, capable of processing both G.U and G.T mispairs. We now show that this latter activity is undetectable in crude extracts of P. aerophilum. However, uracil residues in G.U mispairs, in A.U pairs, and in single-stranded DNA were efficiently removed in these extracts. These activities were assigned to a approximately 22-kDa polypeptide named Pa-UDG (P. aerophilum uracil-DNA glycosylase). The recombinant Pa-UDG protein is highly thermostable and displays a considerable degree of homology to the recently described uracil-DNA glycosylases from Archaeoglobus fulgidus and Thermotoga maritima. Interestingly, neither Pa-MIG nor Pa-UDG was inhibited by UGI, a generic inhibitor of the UNG family of uracil glycosylases. Yet a small fraction of the total uracil processing activity present in crude extracts of P. aerophilum was inhibited by this peptide. This implies that the hyperthermophilic archaeon possesses at least a three-pronged defense against the mutagenic threat of hydrolytic deamination of cytosines in its genomic DNA.

Endonasal and transcanalicular Er:YAG laser dacryocystorhinostomy.

In the last 10 years different types of lasers were used for dacryocystorhinostomy (DCR). Between April 1998 and August 1999, a fibreoptic erbium laser DCR was performed on 12 patients. Eight cases were for a presaccal stenosis and 4 cases for a postsaccal stenosis. An erbium laser with a specially designed handpiece was used endonasaly and transcanaliculary. Preoperative epiphora was present in all patients. Double bicanalicular nasal silicone tubes were placed during surgery in all cases. The 3 cases of postoperative failure included 2 cases of presaccal stenosis and 1 case of the postsaccal group; failure manifested with recurrent epiphora/dacryocystitis; the onset of symptom recurrence varied from 9 weeks to 11 weeks postoperatively. Laser-assisted DCR includes the avoidance of a cutaneous incision, excessive tissue injury, the advantage of short operation time and precision. Suitable indications for the erbium laser are stenoses in the canaliculi, in the sac, but also for bone lacrimal bone cutting.

Separating substrate recognition from base hydrolysis in human thymine DNA glycosylase by mutational analysis.

Human thymine DNA glycosylase (TDG) was discovered as an enzyme that can initiate base excision repair at sites of 5-methylcytosine- or cytosine deamination in DNA by its ability to release thymine or uracil from G.T and G.U mismatches. Crystal structure analysis of an Escherichia coli homologue identified conserved amino acid residues that are critical for its substrate recognition/interaction and base hydrolysis functions. Guided by this revelation, we performed a mutational study of structure function relationships with the human TDG. Substitution of the postulated catalytic site asparagine with alanine (N140A) resulted in an enzyme that bound mismatched substrates but was unable to catalyze base removal. Mutation of Met-269 in a motif with a postulated role in protein-substrate interaction selectively inactivated stable binding of the enzyme to mismatched substrates but not so its glycosylase activity. These results establish that the structure function model postulated for the E. coli enzyme is largely applicable to the human TDG. We further provide evidence for G.U being the preferred substrate of TDG, not only at the mismatch recognition step of the reaction but also in base hydrolysis, and for the importance of stable complementary strand interactions by TDG to compensate for its comparably poor hydrolytic potential.

Identification of hMutLbeta, a heterodimer of hMLH1 and hPMS1.

hMLH1 and hPMS2 function in postreplicative mismatch repair in the form of a heterodimer referred to as hMutLalpha. Tumors or cell lines lacking this factor display mutator phenotypes and microsatellite instability, and mutations in the hMLH1 and hPMS2 genes predispose to hereditary non-polyposis colon cancer. A third MutL homologue, hPMS1, has also been reported to be mutated in one cancer-prone kindred, but the protein encoded by this locus has so far remained without function. We now show that hPMS1 is expressed in human cells and that it interacts with hMLH1 with high affinity to form the heterodimer hMutLbeta. Recombinant hMutLalpha and hMutLbeta, expressed in the baculovirus system, were tested for their activity in an in vitro mismatch repair assay. While hMutLalpha could fully complement extracts of mismatch repair-deficient cell lines lacking hMLH1 or hPMS2, hMutLbeta failed to do so with any of the different substrates tested in this assay. The involvement of the latter factor in postreplicative mismatch repair thus remains to be demonstrated.

Surgical treatment of Zenker's diverticulum: transcutaneous diverticulectomy versus microendoscopic myotomy of the cricopharyngeal muscle with CO2 laser.

In a retrospective study, we analyzed 97 patients who were treated by either transcutaneous diverticulectomy (n = 66) or microendoscopic myotomy of the cricopharyngeal muscle with CO(2) laser (n = 31). Two (6.4%) of 31 patients in the microendoscopic myotomy group had complications, compared with 10 (15%) of 66 patients in the diverticulectomy group. In addition, the complications observed in the microendoscopic myotomy group were less severe than those observed in the transcutaneous diverticulectomy group. The average length of hospitalization was shorter in the microendoscopic myotomy group than in the diverticulectomy group (8 days versus 11.4 days). We conclude that microendoscopic CO(2)-laser myotomy is a less invasive, more precise, and safer procedure, which results in a shortened period of hospitalization and complete relief of symptoms in the vast majority of cases.

Advantages and dangers of erbium laser application in stapedotomy.

Among different types of lasers, the erbium laser exhibits particularly favourable characteristics for ear surgery. Experiments with application of erbium laser pulses to the isolated stapes connected to an inner ear model confirmed that there was virtually no thermal effect to the inner ear liquid and that the border damage zone on the stapes footplate perforation did not exceed 5-10 microm. Erbium laser pulses, however, produce pressure waves due to the explosive ablation of tissue. Pulses of 10 to 17 J/cm2 producing pressure waves between 140 and 160 dB appear to be a limit for clinical application. With these criteria, an in-house built erbium YAG laser with a fiberoptic delivery device was used in 15 patients for stapedotomy. A special microhandpiece, where a zirconium fluoride fiber was connected to a quartz tip, was developed. In addition, three patients had stapedotomy with a commercially available Zeiss (Opmi TwinER) microscope equipped with a micromanipulator-operated erbium laser beam. One year after surgery, the air-bone gap was closed in all patients to within 20 dB between 0.5 and 3 kHz with only minor permanent bone conduction threshold losses (< 20 dB). However, we observed an immediate postoperative middle and high frequency loss of up to 75 dB on bone conduction threshold measurements 2 h after surgery, suggesting an acoustic traumatization by the erbium laser. This threshold shift recovered close to preoperative values within 6 h. These observations prompted us to discontinue the clinical use of erbium laser for stapedotomy until the problem of temporary acoustic traumatization is resolved.

Involvement of nucleotide-excision repair in msh2 pms1-independent mismatch repair.

Nucleotide-excision repair (NER) and mismatch repair (MMR) are prominent examples of highly conserved DNA repair systems which recognize and replace damaged and/or mispaired nucleotides in DNA. In humans, inheritable defects in components of the NER system are associated with severe diseases such as xeroderma pigmentosum (XP) and Cockayne syndrome (CS), whereas inactivation of MMR is accompanied by predisposition to certain types of cancer. In Schizosaccharomyces pombe, the msh2- and pms1-dependent long-patch MMR system efficiently corrects small insertion/deletion loops and all base-base mismatches, except C/C. Up to 70% of C/C mismatches generated in recombination intermediates, and to a lesser extent also other base-base mismatches, are thought to undergo correction by a minor, short-patch excision repair system. We identify here the NER genes rhpl4, swi10 and rad16 as components of this repair pathway and show that they act independently of msh2 and pms1.

Recognition of DNA alterations by the mismatch repair system.

Misincorporation of non-complementary bases by DNA polymerases is a major source of the occurrence of promutagenic base-pairing errors during DNA replication or repair. Base-base mismatches or loops of extra bases can arise which, if left unrepaired, will generate point or frameshift mutations respectively. To counteract this mutagenic potential, organisms have developed a number of elaborate surveillance and repair strategies which co-operate to maintain the integrity of their genomes. An important replication-associated correction function is provided by the post-replicative mismatch repair system. This system is highly conserved among species and appears to be the major pathway for strand-specific elimination of base-base mispairs and short insertion/deletion loops (IDLs), not only during DNA replication, but also in intermediates of homologous recombination. The efficiency of repair of different base-pairing errors in the DNA varies, and appears to depend on multiple factors, such as the physical structure of the mismatch and sequence context effects. These structural aspects of mismatch repair are poorly understood. In contrast, remarkable progress in understanding the biochemical role of error-recognition proteins has been made in the recent past. In eukaryotes, two heterodimers consisting of MutS-homologous proteins have been shown to share the function of mismatch recognition in vivo and in vitro. A first MutS homologue, MSH2, is present in both heterodimers, and the specificity for mismatch recognition is dictated by its association with either of two other MutS homologues: MSH6 for recognition of base-base mismatches and small IDLs, or MSH3 for recognition of IDLs only. Mismatch repair deficiency in cells can arise through mutation, transcriptional silencing or as a result of imbalanced expression of these genes.

Saccharomyces cerevisiae LIF1: a function involved in DNA double-strand break repair related to mammalian XRCC4.

Saccharomyces cerevisiae DNA ligase IV (LIG4) has been shown previously to be involved in non-homologous DNA end joining and meiosis. The homologous mammalian DNA ligase IV interacts with XRCC4, a protein implicated in V(D)J recombination and double-strand break repair. Here, we report the discovery of LIF1, a S.cerevisiae protein that strongly interacts with the C-terminal BRCT domain of yeast LIG4. LIG4 and LIF1 apparently occur as a heterodimer in vivo. LIF1 shares limited sequence homology with mammalian XRCC4. Disruption of the LIF1 gene abolishes the capacity of cells to recircularize transformed linearized plasmids correctly by non-homologous DNA end joining. Loss of LIF1 is also associated with conditional hypersensitivity of cells to ionizing irradiation and with reduced sporulation efficiency. Thus, with respect to their phenotype, lif1 strains are similar to the previously described lig4 mutants. One function of LIF1 is the stabilization of the LIG4 enzyme. The finding of a XRCC4 homologue in S.cerevisiae now allows for mutational analyses of structure-function relationships in XRCC4-like proteins to define their role in DNA double-strand break repair.

A newly identified DNA ligase of Saccharomyces cerevisiae involved in RAD52-independent repair of DNA double-strand breaks.

Eukaryotic DNA ligases are ATP-dependent DNA strand-joining enzymes that participate in DNA replication, repair, and recombination. Whereas mammalian cells contain several different DNA ligases, encoded by at least three distinct genes, only one DNA ligase has been detected previously in either budding yeast or fission yeast. Here, we describe a newly identified nonessential Saccharomyces cerevisiae gene that encodes a DNA ligase distinct from the CDC9 gene product. This DNA ligase shares significant amino acid sequence homology with human DNA ligase IV; accordingly, we designate the yeast gene LIG4. Recombinant LIG4 protein forms a covalent enzyme-AMP complex and can join a DNA single-strand break in a DNA/RNA hybrid duplex, the preferred substrate in vitro. Disruption of the LIG4 gene causes only marginally increased cellular sensitivity to several DNA damaging agents, and does not further sensitize cdc9 or rad52 mutant cells. In contrast, lig4 mutant cells have a 1000-fold reduced capacity for correct recircularization of linearized plasmids by illegitimate end-joining after transformation. Moreover, homozygous lig4 mutant diploids sporulate less efficiently than isogenic wild-type cells, and show retarded progression through meiotic prophase I. Spore viability is normal, but lig4 mutants appear to produce a higher proportion of tetrads with only three viable spores. The mutant phenotypes are consistent with functions of LIG4 in an illegitimate DNA end-joining pathway and ensuring efficient meiosis.

Mismatch repair in Schizosaccharomyces pombe requires the mutL homologous gene pms1: molecular cloning and functional analysis.

Homologues of the bacterial mutS and mutL genes involved in DNA mismatch repair have been found in organisms from bacteria to humans. Here, we describe the structure and function of a newly identified Schizosaccharomyces pombe that encodes a predicted amino acid sequence of 794 residues with a high degree of homology to MutL related proteins. On the basis of its closer relationship to the eukaryotic "PMS" genes than to the "MLH" genes, we have designated the S. pombe homologue pms1. Disruption of the pms1 gene causes a significant increase of spontaneous mutagenesis as documented by reversion rate measurements. Tetrad analyses of crosses homozygous for the pms1 mutation reveal a reduction of spore viability from > 92% to 80% associated with a low proportion (approximately 50%) of meioses producing four viable spores and a significant, allele-dependent increase of the level of post-meiotic segregation of genetic marker allele pairs. The mutant phenotypes are consistent with a general function of pms1 in correction of mismatched base pairs arising as a consequence of DNA polymerase errors during DNA synthesis, or of hybrid DNA formation between homologous but not perfectly complementary DNA strands during meiotic recombination.

Regulation of DNA metabolic enzymes upon induction of preB cell development and V(D)J recombination: up-regulation of DNA polymerase delta.

Withdrawal of interleukin-7 from cultured murine preB lymphocytes induces cell differentiation including V(D)J immunoglobulin gene rearrangements and cell cycle arrest. Advanced steps of the V(D)J recombination reaction involve processing of coding ends by several largely unidentified DNA metabolic enzymes. We have analyzed expression and activity of DNA polymerases alpha, beta, delta and epsilon, proliferating cell nuclear antigen (PCNA), topoisomerases I and II, terminal deoxynucleotidyl transferase (TdT) and DNA ligases I, III and IV upon induction of preB cell differentiation. Despite the immediate arrest of cell proliferation, DNA polymerase delta protein levels remained unchanged for approximately 2 days and its activity was up-regulated several-fold, while PCNA was continuously present. Activity of DNA polymerases alpha,beta and epsilon decreased. Expression and activity of DNA ligase I were drastically reduced, while those of DNA ligases III and IV remained virtually constant. No changes in DNA topoisomerases I or II expression and activity occurred and TdT expression was moderately increased early after induction. Our results render DNA polymerase delta a likely candidate acting in DNA synthesis related to V(D)J recombination in lymphocytes.

Repair and processing events at DNA ends.

Cell nuclei contain several abundant enzymes that bind rapidly and avidly to exposed termini of DNA. The properties and physiological roles of such factors are described; they include poly (ADP-ribose) polymerase, DNA-dependent protein kinase, several DNA ligases and excision-repair enzymes. Telomeres normally seem shielded from these activities by telomere-binding proteins. If incomplete protection of telomeres occurred, the functions of the DNA end-specific enzymes would be relevant for processing of telomeres. This could include alternative pathways for telomere propagation in telomerase-negative cells.

Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein.

Repair of a uracil-guanine base pair in DNA has been reconstituted with the recombinant human proteins uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta and DNA ligase III. The XRCC1 protein, which is known to bind DNA ligase III, is not absolutely required for the reaction but suppresses strand displacement by DNA polymerase beta, allowing for more efficient ligation after filling of a single nucleotide patch. We show that XRCC1 interacts directly with DNA polymerase beta using far Western blotting, affinity precipitation and yeast two-hybrid analyses. In addition, a complex formed between DNA polymerase beta and a double-stranded oligonucleotide containing an incised abasic site was supershifted by XRCC1 in a gel retardation assay. The region of interaction with DNA polymerase beta is located within residues 84-183 in the N-terminal half of the XRCC1 protein, whereas the C-terminal region of XRCC1 is involved in binding DNA ligase III. These data indicate that XRCC1, which has no known catalytic activity, might serve as a scaffold protein during base excision-repair. DNA strand displacement and excessive gap filling during DNA repair were observed in cell-free extracts of an XRCC1-deficient mutant cell line, in agreement with the results from the reconstituted system.

Molecular cloning and expression of human cDNAs encoding a novel DNA ligase IV and DNA ligase III, an enzyme active in DNA repair and recombination.

Three distinct DNA ligases, I to III, have been found previously in mammalian cells, but a cloned cDNA has been identified only for DNA ligase I, an essential enzyme active in DNA replication. A short peptide sequence conserved close to the C terminus of all known eukaryotic DNA ligases was used to search for additional homologous sequences in human cDNA libraries. Two different incomplete cDNA clones that showed partial homology to the conserved peptide were identified. Full-length cDNAs were obtained and expressed by in vitro transcription and translation. The 103-kDa product of one cDNA clone formed a characteristic complex with the XRCC1 DNA repair protein and was identical with the previously described DNA ligase III. DNA ligase III appears closely related to the smaller DNA ligase II. The 96-kDa in vitro translation product of the second cDNA clone was also shown to be an ATP-dependent DNA ligase. A fourth DNA ligase (DNA ligase IV) has been purified from human cells and shown to be identical to the 96-kDa DNA ligase by unique agreement between mass spectrometry data on tryptic peptides from the purified enzyme and the predicted open reading frame of the cloned cDNA. The amino acid sequences of DNA ligases III and IV share a related active-site motif and several short regions of homology with DNA ligase I, other DNA ligases, and RNA capping enzymes. DNA ligases III and IV are encoded by distinct genes located on human chromosomes 17q11.2-12 and 13q33-34, respectively.