Roles of the DNA mismatch repair and nucleotide excision repair proteins during meiosis

Numerous proteins are involved in the nucleotide excision repair (NER) and DNA mismatch repair (MMR) pathways. The function and specificity of these proteins during the mitotic cell cycle has been actively investigated, in large part due to the involvement of these systems in human diseases. In contrast, comparatively little is known about their functioning during meiosis. At least three repair pathways operate during meiosis in the yeast Saccharomyces cerevisiae to repair mismatches that occur as a consequence of heteroduplex formation in recombination. The first pathway is similar to the one acting during postreplicative mismatch repair in mitotically dividing cells, while two pathways are responsible for the repair of large loops during meiosis, using proteins from MMR and NER systems. Some MMR proteins also help prevent recombination between diverged sequences during meiosis, and act late in recombination to affect the resolution of crossovers. This review will discuss the current status of DNA mismatch repair and nucleotide excision repair proteins during meiosis, especially in the yeast S. cerevisiae. Received 21 September 1998; received after revision 23 November 1998; accepted 23 November 1998     

Homologous recombination-mediated repair of DNA double-strand breaks operates in mammalian mitochondria

Mitochondrial DNA is frequently exposed to oxidative damage, as compared to nuclear DNA. Previously, we have shown that while microhomology-mediated end joining can account for DNA deletions in mitochondria, classical nonhomologous DNA end joining, the predominant double-strand break (DSB) repair pathway in nucleus, is undetectable. In the present study, we investigated the presence of homologous recombination (HR) in mitochondria to maintain its genomic integrity. Biochemical studies revealed that HR-mediated repair of DSBs is more efficient in the mitochondria of testes as compared to that of brain, kidney and spleen. Interestingly, a significant increase in the efficiency of HR was observed when a DSB was introduced. Analyses of the clones suggest that most of the recombinants were generated through reciprocal exchange, while ~ 30% of recombinants were due to gene conversion in testicular extracts. Colocalization and immunoblotting studies showed the presence of RAD51 and MRN complex proteins in the mitochondria and immunodepletion of MRE11, RAD51 or NIBRIN suppressed the HR-mediated repair. Thus, our results reveal importance of homologous recombination in the maintenance of mitochondrial genome stability.     

Sea urchin embryo as a model for analysis of the signaling pathways linking DNA damage checkpoint,DNA repair and apoptosis

DNA integrity checkpoint control was studied in the sea urchin early embryo. Treatment of the embryos with genotoxic agents such as methyl methanesulfonate (MMS) or bleomycin induced the activation of a cell cycle checkpoint as evidenced by the occurrence of a delay or an arrest in the division of the embryos and an inhibition of CDK1/cyclin B activating dephosphorylation. The genotoxic treatment was shown to induce DNA damage that depended on the genotoxic concentration and was correlated with the observed cell cycle delay. At low genotoxic concentrations, embryos were able to repair the DNA damage and recover from checkpoint arrest, whereas at high doses they underwent morphological and biochemical changes characteristic of apoptosis. Finally, extracts prepared from embryos were found to be capable of supporting DNA repair in vitro upon incubation with oligonucleotides mimicking damage. Taken together, our results demonstrate that sea urchin early embryos contain fully functional and activatable DNA damage checkpoints. Sea urchin embryos are discussed as a promising model to study the signaling pathways of cell cycle checkpoint, DNA repair and apoptosis, which upon deregulation play a significant role in the origin of cancer. Received 10 April 2007; accepted 23 April 2007     

Base excision DNA repair

DNA repair is a collection of several multienzyme, multistep processes keeping the cellular genome intact against genotoxic insults. One of these processes is base excision repair, which deals with the most ubiquitous lesions in DNA: oxidative base damage, alkylation, deamination, sites of base loss and single-strand breaks, etc. Individual enzymes acting in base excision repair have been identified. The recent years were marked with many advances in understanding of their structure and many interactions that make base excision repair a functional, versatile system. This review describes the current knowledge of structural biology and biochemistry of individual steps of base excision repair, several subpathways of the common base excision repair pathway, and interactions of the repair process with other cellular processes.     

Host cell reactivation of ozone-treated T3 bacteriophage by different strains ofEscherichia coli

Summary Host cell reactivation capacity for ozone T3 phage was determined for differentE. coli strains deficient in one or more of the DNA repair mechanisms. The results indicate that DNA polymerase I appears to play a key role in the repair of damage produced on the DNA by ozone while thelexA gene product seems to play a minor one.This research was sponsored by the National Sciences and Engineering Council of Canada. One of us (PLH) acknowledges a scholarship from the NSERCof Canada.     

Intron creation and DNA repair

The genesis of the exon–intron patterns of eukaryotic genes persists as one of the most enigmatic questions in molecular genetics. In particular, the origin and mechanisms responsible for creation of spliceosomal introns have remained controversial. Now the issue appears to have taken a turn. The formation of novel introns in eukaryotes, including some vertebrate lineages, is not as rare as commonly assumed. Moreover, introns appear to have been gained in parallel at closely spaced sites and even repeatedly at the same position. Based on these discoveries, novel hypotheses of intron creation have been developed. The new concepts posit that DNA repair processes are a major source of intron formation. Here, after summarizing the current views of intron gain mechanisms, I review findings in support of the DNA repair hypothesis that provides a global mechanistic scenario for intron creation. Some implications on our perception of the mosaic structure of eukaryotic genes are also discussed.     

Polarity of meiotic gene conversion in fungi: Contrasting views

The frequency of meiotic gene conversion often varies linearly from one end of the gene to the other. This phenomenon has been called polarity. In this review, we will primarily discuss studies of polarity that have been done in the yeastSaccharomyces cerevisiae (ARG4 and HIS4 loci) and inAscobolus (b2 locus) with an emphasis on possible mechanisms. The genetic and physical data obtained at these hotspots of recombination strongly suggests that the formation of a polarity gradient reflects both the frequency of heteroduplex formation and the processing of this recombination intermediate by mismatch-repair-dependent processes.This article is dedicated to the memory of Seymour Fogel.     

Participation of DNA repair in the response to 5-fluorouracil

The anti-metabolite 5-fluorouracil (5-FU) is employed clinically to manage solid tumors including colorectal and breast cancer. Intracellular metabolites of 5-FU can exert cytotoxic effects via inhibition of thymidylate synthetase, or through incorporation into RNA and DNA, events that ultimately activate apoptosis. In this review, we cover the current data implicating DNA repair processes in cellular responsiveness to 5-FU treatment. Evidence points to roles for base excision repair (BER) and mismatch repair (MMR). However, mechanistic details remain unexplained, and other pathways have not been exhaustively interrogated. Homologous recombination is of particular interest, because it resolves unrepaired DNA intermediates not properly dealt with by BER or MMR. Furthermore, crosstalk among DNA repair pathways and S-phase checkpoint signaling has not been examined. Ongoing efforts aim to design approaches and reagents that (i) approximate repair capacity and (ii) mediate strategic regulation of DNA repair in order to improve the efficacy of current anticancer treatments. Received 08 September 2008; received after revision 25 September 2008; accepted 03 October 2008     

Epigenetic mechanisms in mammals

DNA and histone methylation are linked and subjected to mitotic inheritance in mammals. Yet how methylation is propagated and maintained between successive cell divisions is not fully understood. A series of enzyme families that can add methylation marks to cytosine nucleobases, and lysine and arginine amino acid residues has been discovered. Apart from methyltransferases, there are also histone modification enzymes and accessory proteins, which can facilitate and/or target epigenetic marks. Several lysine and arginine demethylases have been discovered recently, and the presence of an active DNA demethylase is speculated in mammalian cells. A mammalian methyl DNA binding protein MBD2 and de novo DNA methyltransferase DNMT3A and DNMT3B are shown experimentally to possess DNA demethylase activity. Thus, complex mammalian epigenetic mechanisms appear to be dynamic yet reversible along with a well-choreographed set of events that take place during mammalian development.     

Alkyltransferase-like proteins: molecular switches between DNA repair pathways

Alkyltransferase-like proteins (ATLs) play a role in the protection of cells from the biological effects of DNA alkylation damage. Although ATLs share functional motifs with the DNA repair protein and cancer chemotherapy target O ⁶-alkylguanine-DNA alkyltransferase, they lack the reactive cysteine residue required for alkyltransferase activity, so its mechanism for cell protection was previously unknown. Here we review recent advances in unraveling the enigmatic cellular protection provided by ATLs against the deleterious effects of DNA alkylation damage. We discuss exciting new evidence that ATLs aid in the repair of DNA O ⁶-alkylguanine lesions through a novel repair cross-talk between DNA-alkylation base damage responses and the DNA nucleotide excision repair pathway.     

The search for the right partner: Homologous pairing and DNA strand exchange proteins in eukaryotes

Finding the right partner is a central problem in homologous recombination. Common to all models for general recombination is a homologous pairing and DNA strand exchange step. In prokaryotes this process has mainly been studied with the RecA protein ofEscherichia coli. Two approaches have been used to find homologous pairing and DNA strand exchange proteins in eukaryotes. A biochemical approach has resulted in numerous proteins from various organisms. Almost all of these proteins are biochemically fundamentally different from RecA. The in vivo role of these proteins is largely not understood. A molecular-genetical approach has identified structural homologs to theE. coli RecA protein in the yeastSaccharomyces cerevisiae and subsequently in other organisms including other fungi, mammals, birds, and plants. The biochemistry of the eukaryotic RecA homologs is largely unsolved. For the fungal RecA homologs (S. cerevisiae RAD51, RAD55, RAD57, DMC1; Schizosaccharomyces pombe rad51; Neurospora crassa mei3) a role in homologous recombination and recombinational repair is evident. Besides recombination, homologous pairing proteins might be involved in other cellular processes like chromosome pairing or gene inactivation.     

Biochemical aspects of radiation biology

Summary In order to analyze the mechanisms of biological radiation effects, the events after radiation energy absorption in irradiated organisms have to be studied by physico-chemical and biochemical methods. The radiation effects in vitro on biomolecules, especially DNA, are described, as well as their alterations in irradiated cells. Whereas in vitro, in aqueous solution, predominantly OH radicals are effective and lead to damage in single moieties of the DNA, in vivo the direct absorption of radiation energy leads to locally multiply-damaged sites, which produce DNA double-strand breaks and locally denatured regions. DNA damage will be repaired in irradiated cells. Error free repair leads to the original nucleotide sequence in the genome by excision or by recombination. Error prone repair (mutagenic repair), leads to mutation. However, the biochemistry of these processes, regulated by a number of genes, is poorly understood. In addition, more complex reactions, such as gene amplification and transposition of mobile gene elements, are responsible for mutation or malignant transformation.     

Satellite DNA from the beetleTenebrio molitor

Summary A 142 base-pair satellite DNA from the mealworm beetle,Tenebrio molitor, has been cloned and sequenced. The satellite DNA is revealed by making a restriction digest of genomic DNA with either EcoRI or Hinfl, and constitutes approximately 49% of the genomic DNA. The presence of huge amounts of satellite DNA correlates well with the prominent blocks of heterochromatin found in tenebrionid beetles. A similar restriction digest ofXanthogaleruca luteola genomic DNA does not release a prominent satellite component.     

Implication of the VRK1 chromatin kinase in the signaling responses to DNA damage: a therapeutic target?

DNA damage causes a local distortion of chromatin that triggers the sequential processes that participate in specific DNA repair mechanisms. This initiation of the repair response requires the involvement of a protein whose activity can be regulated by histones. Kinases are candidates to regulate and coordinate the connection between a locally altered chromatin and the response initiating signals that lead to identification of the type of lesion and the sequential steps required in specific DNA damage responses (DDR). This initiating kinase must be located in chromatin, and be activated independently of the type of DNA damage. We review the contribution of the Ser-Thr vaccinia-related kinase 1 (VRK1) chromatin kinase as a new player in the signaling of DNA damage responses, at chromatin and cellular levels, and its potential as a new therapeutic target in oncology. VRK1 is involved in the regulation of histone modifications, such as histone phosphorylation and acetylation, and in the formation of γH2AX, NBS1 and 53BP1 foci induced in DDR. Induction of DNA damage by chemotherapy or radiation is a mainstay of cancer treatment. Therefore, novel treatments can be targeted to proteins implicated in the regulation of DDR, rather than by directly causing DNA damage.     

Gene inactivation triggered by recognition between DNA repeats

This chapter focuses on phenomena of gene inactivation resulting from the presence of repeated gene copies within the genome of plants and fungi, and on their possible relationships to homologous DNA-DNA interactions. Emphasis is given to two related premeiotic processes: Methylation Induced Premeiotically (MIP) and Repeat-Induced Point mutation (RIP) which take place in the fungiAscobolus immersus andNeurospora crassa, respectively. The relationships between these processes and genetic recombination are discussed.