Serge Boiteux

Formamidopyrimidine-DNA glycosylase of Escherichia coli: cloning and sequencing of the fpg structural gene and overproduction of the protein

An Escherichia coli genomic library composed of large DNA fragments (10‐15 kb) was constructed using the plasmid pBR322 as vector. From it 700 clones were individually screened for increased excision of the ring‐opened form of N7‐methylguanine (2‐6‐diamino‐4‐hydroxy‐5N‐methyl‐formamidopyrimidine) or Fapy. One clone overproduced the Fapy‐DNA glycosylase activity by a factor of 10‐fold as compared with the wild‐type strain. The Fapy‐DNA glycosylase overproducer character was associated with a 15‐kb recombinant plasmid (pFPG10). After subcloning a 1.4‐kb fragment which contained the Fapy‐DNA glycosylase gene (fpg+) was inserted in the plasmids pUC18 and pUC19 yielding pFPG50 and pFPG60 respectively. The cells harbouring pFPG60 displayed a 50‐ to 100‐fold increase in glycosylase activity and overexpressed a 31‐kd protein. From these cells the Fapy‐DNA glycosylase was purified to apparent physical homogeneity as evidenced by a single protein band at 31 kd on SDS‐polyacrylamide gels. The amino acid composition of the protein and the amino acid sequence deduced from the nucleotide sequence demonstrate that the cloned fragment contains the structural gene coding for the Fapy‐DNA glycosylase. The nucleotide sequence of the fpg gene is composed of 809 base pairs and codes for a protein of 269 amino acids with a calculated mol. wt of 30.2 kd.

Inactivation of OGG1 increases the incidence of G · C→T · A transversions in Saccharomyces cerevisiae : evidence for endogenous oxidative damage to DNA in eukaryotic cells

Abstract The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. To investigate the biological role of the OGG1 gene, mutants were constructed by partial deletion of the coding sequence and insertion of marker genes, yielding ogg1::TRP1 and ogg1::URA3 mutant strains. The disruption of the OGG1 gene does not compromise the viability of haploid cells, therefore it is not an essential gene. The capacity to repair 8-OxoG has been measured in cell-free extracts of wild-type and ogg1 strains using a 34mer DNA fragment containing a single 8-OxoG residue paired with a cytosine (8-OxoG/C) as a substrate. Cell-free extracts of the wild-type strain efficiently cleave the 8-OxoG-containing strand of the 8-OxoG/C duplex. In contrast, cell-free extracts of the Ogg1-deficient strain have no detectable activity that can cleave the 8-OxoG/C duplex. The biological properties of the ogg1 mutant have also been investigated. The results show that the ogg1 disruptant is not hypersensitive to DNA-damaging agents such as ultraviolet light at 254u2009nm, hydrogen peroxide or methyl methanesulfonate. However, the ogg1 mutant exhibits a mutator phenotype. When compared to those of a wild-type strain, the frequencies of mutation to canavanine resistance (CanR) and reversion to Lys+ are sevenfold and tenfold higher for the ogg1 mutant strain, respectively. Moreover, using a specific tester system, we show that the Ogg1-deficient strain displays a 50-fold increase in spontaneously occurring Gu2009·u2009C→Tu2009·u2009A transversions compared to the wild-type strain. The five other base substitution events are not affected by the disruption of the OGG1 gene. These results strongly suggest that endogeneous reactive oxygen species cause DNA damage and that the excision of 8-OxoG catalyzed by the Ogg1 protein contributes to the maintenance of genetic stability in S. cerevisiae.

Base excision repair of 8-hydroxyguanine protects DNA from endogenous oxidative stress.

A particularly important stress for all cells is the one produced by reactive oxygen species (ROS) that are formed as a byproduct of endogenous metabolism or the exposure to environmental oxidizing agents. An oxidatively damaged guanine, 8-hydroxyguanine (8-OH-G), is abundantly produced in DNA exposed to ROS. The biological relevance of this kind of DNA damage has been unveiled by the study of two mutator genes in E. coli, fpg and mutY. Both genes code for DNA glycosylases that cooperate to prevent the mutagenic effects of 8-OH-G. Inactivation of any of those two genes leads to a spontaneous mutator phenotype characterized by the exclusive increase in G:C to T:A transversions. In the simple eukaryote Saccharomyces cerevisiae, the OGG1 gene encodes an 8-OH-G DNA glycosylase which is the functional homolog of the bacterial fpg gene product. Moreover, the inactivation of OGG1 in yeast creates a mutator phenotype that is also specific for the generation of G:C to T:A transversions. The presence of such system in mammals has been confirmed by the cloning of the OGG1 gene coding for a human homolog of the yeast enzyme. Human cells also possess a MutY homolog encoded by the MYH gene. Analysis of the human OGG1 gene and its transcripts in normal and tumoral tissues reveals alternative splicing, polymorphisms and somatic mutations. The aim of this review is to summarize recent findings dealing with the biochemical properties and the biological functions of 8-OH-G DNA glycosylases in bacterial, yeast, insect and mammalian cells. These results point to 8-OH-G as an endogenous source of mutations and to its likely involvement in the process of carcinogenesis.

Temporally and Biochemically Distinct Activities of Exo1 during Meiosis: Double-Strand Break Resection and Resolution of Double Holliday Junctions

The Rad2/XPG family nuclease, Exo1, functions inxa0a variety of DNA repair pathways. During meiosis, Exo1 promotes crossover recombination and thereby facilitates chromosome segregation at the first division. Meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs). Nucleolytic resection of DSBs generates long 3 single-strand tails that undergo strand exchange with a homologous chromosome to form joint molecule (JM) intermediates. We show that meiotic DSB resection is dramatically reduced in exo1Δ mutants and test the idea that Exo1-catalyzed resection promotes crossing over by facilitating formation of crossover-specific JMs called double Holliday junctions (dHJs). Contrary to this idea, dHJs form at wild-type levels in exo1Δ mutants, implying that Exo1 has a second function that promotes resolution of dHJs into crossovers. Surprisingly, the dHJ resolution function of Exo1 is independent of its nuclease activities but requires interaction with the putative endonuclease complex, Mlh1-Mlh3. Thus, the DSB resection and procrossover functions of Exo1 during meiosis involve temporally and biochemically distinct activities.

Endogenous DNA abasic sites cause cell death in the absence of Apn1, Apn2 and Rad1/Rad10 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, mutations in APN1, APN2 and either RAD1 or RAD10 genes are synthetic lethal. In fact, apn1 apn2 rad1 triple mutants can form microcolonies of ∼300 cells. Expression of Nfo, the bacterial homologue of Apn1, suppresses the lethality. Turning off the expression of Nfo induces G2/M cell cycle arrest in an apn1 apn2 rad1 triple mutant. The activation of this checkpoint is RAD9 dependent and allows residual DNA repair. The Mus81/Mms4 complex was identified as one of these back‐up repair activities. Furthermore, inactivation of Ntg1, Ntg2 and Ogg1 DNA N‐glycosylase/AP lyases in the apn1 apn2 rad1 background delayed lethality, allowing the formation of minicolonies of ∼105 cells. These results demonstrate that, under physiological conditions, endogenous DNA damage causes death in cells deficient in Apn1, Apn2 and Rad1/Rad10 proteins. We propose a model in which endogenous DNA abasic sites are converted into 3′‐blocked single‐strand breaks (SSBs) by DNA N‐glycosylases/AP lyases. Therefore, we suggest that the essential and overlapping function of Apn1, Apn2, Rad1/Rad10 and Mus81/Mms4 is to repair 3′‐blocked SSBs using their 3′‐phosphodiesterase activity or their 3′‐flap endonuclease activity, respectively.

Characterization of the hOGG1 promoter and its expression during the cell cycle.

The human OGG1 gene codes for a 38kD protein with an antimutator activity related to its capacity to excise the mutagenic base 8-OH-Guanine from DNA. Mutant forms of this gene have been found in lung and kidney tumors. The determination of the start of transcription allowed the definition of the promoter sequences for the gene. By transient transfection and a luciferase reporter assay a 135 base pair region immediately upstream of the transcription start is shown to have full promoter activity. Two CpG islands and an Alu repeat were identified within the promoter and the 5 sequences of the transcribed region. The lack of TATA or CAAT boxes suggests that OGG1 is a housekeeping gene. Consistently, its expression, measured as the transcription from the promoter or as the enzymatic activity in cultured fibroblast cell lines, does not vary during the cell cycle.

DNA Repair Mechanisms and the Bypass of DNA Damage in Saccharomyces cerevisiae

DNA repair mechanisms are critical for maintaining the integrity of genomic DNA, and their loss is associated with cancer predisposition syndromes. Studies in Saccharomyces cerevisiae have played a central role in elucidating the highly conserved mechanisms that promote eukaryotic genome stability. This review will focus on repair mechanisms that involve excision of a single strand from duplex DNA with the intact, complementary strand serving as a template to fill the resulting gap. These mechanisms are of two general types: those that remove damage from DNA and those that repair errors made during DNA synthesis. The major DNA-damage repair pathways are base excision repair and nucleotide excision repair, which, in the most simple terms, are distinguished by the extent of single-strand DNA removed together with the lesion. Mistakes made by DNA polymerases are corrected by the mismatch repair pathway, which also corrects mismatches generated when single strands of non-identical duplexes are exchanged during homologous recombination. In addition to the true repair pathways, the postreplication repair pathway allows lesions or structural aberrations that block replicative DNA polymerases to be tolerated. There are two bypass mechanisms: an error-free mechanism that involves a switch to an undamaged template for synthesis past the lesion and an error-prone mechanism that utilizes specialized translesion synthesis DNA polymerases to directly synthesize DNA across the lesion. A high level of functional redundancy exists among the pathways that deal with lesions, which minimizes the detrimental effects of endogenous and exogenous DNA damage.

Crystal structure of the Lactococcus lactis formamidopyrimidine-DNA glycosylase bound to an abasic site analogue-containing DNA

The formamidopyrimidine‐DNA glycosylase (Fpg, MutM) is a bifunctional base excision repair enzyme (DNA glycosylase/AP lyase) that removes a wide range of oxidized purines, such as 8‐oxoguanine and imidazole ring‐opened purines, from oxidatively damaged DNA. The structure of a non‐covalent complex between the Lactoccocus lactis Fpg and a 1,3‐propanediol (Pr) abasic site analogue‐containing DNA has been solved. Through an asymmetric interaction along the damaged strand and the intercalation of the triad (M75/R109/F111), Fpg pushes out the Pr site from the DNA double helix, recognizing the cytosine opposite the lesion and inducing a 60° bend of the DNA. The specific recognition of this cytosine provides some structural basis for understanding the divergence between Fpg and its structural homologue endo nuclease VIII towards their substrate specificities. In addition, the modelling of the 8‐oxoguanine residue allows us to define an enzyme pocket that may accommodate the extrahelical oxidized base.

Repair of 8-oxoguanine in Saccharomyces cerevisiae: interplay of DNA repair and replication mechanisms.

8-Oxo-7,8-dihydroguanine (8-oxoG) is produced abundantly in DNA exposed to free radicals and reactive oxygen species. The biological relevance of 8-oxoG has been unveiled by the study of two mutator genes in Escherichia coli, fpg, and mutY. Both genes code for DNA N-glycosylases that cooperate to prevent the mutagenic effects of 8-oxoG in DNA. In Saccharomyces cerevisiae, the OGG1 gene encodes a DNA N-glycosylase/AP lyase, which is the functional homologue of the bacterial fpg gene product. The inactivation of OGG1 in yeast creates a mutator phenotype that is specific for the generation of GC to TA transversions. In yeast, nucleotide excision repair (NER) also contributes to the release of 8-oxoG in damaged DNA. Furthermore, mismatch repair (MMR) mediated by MSH2/MSH6/MLH1 plays a major role in the prevention of the mutagenic effect of 8-oxoG. Indeed, MMR acts as the functional homologue of the MutY protein of E. coli, excising the adenine incorporated opposite 8-oxoG. Finally, the efficient and accurate replication of 8-oxoG by the yeast DNA polymerase eta also prevents 8-oxoG-induced mutagenesis. The aim of this review is to summarize recent literature dealing with the replication and repair of 8-oxoG in Saccharomyces cerevisiae, which can be used as a paradigm for DNA repair in eukaryotes.

REPAIR OF OXIDIZED DNA BASES IN THE YEAST SACCHAROMYCES CEREVISIAE

An essential requirement for all organisms is to maintain its genomic integrity. Failure to do so, in multicellular organisms such as man, can lead to degenerative pathologies such as cancer and aging. Indeed, a very low spontaneous mutation rate is observed in eukaryotes, suggesting either an inherent stability of the genome or efficient DNA repair mechanisms. In fact, DNA is subjected to unceasing attacks by a variety of endogenous and environmental reactive chemical species yielding a multiplicity of DNA damage, the deleterious action of which is counteracted by efficient repair enzymes. Reactive oxygen species formed in cell as by-products of normal metabolism are probably the major source of endogenous DNA damage. Amongst oxidative damage, base modifications constitute an important class of lesions whose lethal or mutagenic action has been established. Oxidatively damaged DNA bases are mostly repaired by the base excision repair pathway (BER) in prokaryotes and eukaryotes. However, the nucleotide excision repair pathway (NER) may also play a role in the repair of some oxidized bases in DNA. Here, we describe repair pathways implicated in the removal of oxidized bases in Saccharomyces cerevisiae. Yeast is a simple organism that can be used as a paradigm for DNA repair in all eukaryotic cells. S cerevisiae possesses three DNA glycosylases that catalyze the excision of oxidized bases from damaged DNA: the Ogg1, Ntg1 and Ntg2 proteins. The aim of this review is to summarize recent findings dealing with the formation, the biological consequences and the repair of oxidized DNA bases in S cerevisiae.