Stéphanie Marsin

Role of XRCC1 in the Coordination and Stimulation of Oxidative DNA Damage Repair Initiated by the DNA Glycosylase hOGG1

XRCC1 participates in DNA single strand break and base excision repair (BER) to preserve genetic stability in mammalian cells. XRCC1 participation in these pathways is mediated by its interactions with several of the acting enzymes. Here, we report that XRCC1 interacts physically and functionally with hOGG1, the human DNA glycosylase that initiates the repair by BER of the mutagenic oxidized base 8-oxoguanine. This interaction leads to a 2- to 3-fold stimulation of the DNA glycosylase activity of hOGG1. XRCC1 stimulates the formation of the hOGG1 Schiff-base DNA intermediate without interfering with the endonuclease activity of APE1, the second enzyme in the pathway. On the contrary, the stimulation in the appearance of the incision product seems to reflect the addition of the effects of XRCC1 on the two first enzymes of the pathway. The data presented support a model by which XRCC1 will pass on the DNA intermediate from hOGG1 to the endonuclease APE1. This results in an acceleration of the overall repair process of oxidized purines to yield an APE1-cleaved abasic site, which can be used as a substrate by DNA polymerase β. More importantly, the results unveil a highly coordinated mechanism by which XRCC1, through its multiple protein-protein interactions, extends its orchestrating role from the base excision step to the resealing of the repaired DNA strand.

Redox regulation of human OGG1 activity in response to cellular oxidative stress.

ABSTRACT 8-Oxoguanine (8-oxoG), a common and mutagenic form of oxidized guanine in DNA, is eliminated mainly through base excision repair. In human cells its repair is initiated by human OGG1 (hOGG1), an 8-oxoG DNA glycosylase. We investigated the effects of an acute cadmium exposure of human lymphoblastoid cells on the activity of hOGG1. We show that coinciding with alteration of the redox cellular status, the 8-oxoG DNA glycosylase activity of hOGG1 was nearly completely inhibited. However, the hOGG1 activity returned to normal levels once the redox cellular status was normalized. In vitro, the activity of purified hOGG1 was abolished by cadmium and could not be recovered by EDTA. In cells, however, the reversible inactivation of OGG1 activity by cadmium was strictly associated with reversible oxidation of the protein. Moreover, the 8-oxoG DNA glycosylase activity of purified OGG1 and that from crude extracts were modulated by cysteine-modifying agents. Oxidation of OGG1 by the thiol oxidant diamide led to inhibition of the activity and a protein migration pattern similar to that seen in cadmium-treated cells. These results suggest that cadmium inhibits hOGG1 activity mainly by indirect oxidation of critical cysteine residues and that excretion of the metal from the cells leads to normalization of the redox cell status and restoration of an active hOGG1. The results presented here unveil a novel redox-dependent mechanism for the regulation of OGG1 activity.

Unveiling Novel RecO Distant Orthologues Involved in Homologous Recombination

The generation of a RecA filament on single-stranded DNA is a critical step in homologous recombination. Two main pathways leading to the formation of the nucleofilament have been identified in bacteria, based on the protein complexes mediating RecA loading: RecBCD (AddAB) and RecFOR. Many bacterial species seem to lack some of the components involved in these complexes. The current annotation of the Helicobacter pylori genome suggests that this highly diverse bacterial pathogen has a reduced set of recombination mediator proteins. While it is now clear that homologous recombination plays a critical role in generating H. pylori diversity by allowing genomic DNA rearrangements and integration through transformation of exogenous DNA into the chromosome, no complete mediator complex is deduced from the sequence of its genome. Here we show by bioinformatics analysis the presence of a RecO remote orthologue that allowed the identification of a new set of RecO proteins present in all bacterial species where a RecR but not RecO was previously identified. HpRecO shares less than 15% identity with previously characterized homologues. Genetic dissection of recombination pathways shows that this novel RecO and the remote RecB homologue present in H. pylori are functional in repair and in RecA-dependent intrachromosomal recombination, defining two initiation pathways with little overlap. We found, however, that neither RecOR nor RecB contributes to transformation, suggesting the presence of a third, specialized, RecA-dependent pathway responsible for the integration of transforming DNA into the chromosome of this naturally competent bacteria. These results provide insight into the mechanisms that this successful pathogen uses to generate genetic diversity and adapt to changing environments and new hosts.

A rolling circle replication initiator protein with a nucleotidyl‐transferase activity encoded by the plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi presents similarities to plasmids from the pC194 family that replicate by the rolling circle mechanism. These plasmids encode a replication initiator protein, which activates the replication origin by nicking one of the two DNA strands. The gene encoding the putative Rep protein of pGT5 (Rep75) has been cloned and overexpressed in Escherichia coli, and the recombinant protein has been purified to homogeneity. Rep75 exhibits a highly thermophilic nicking‐closing activity in vitro on single‐stranded oligonucleotides containing the putative double‐stranded replication origin sequence of pGT5. Gel shift analyses on single‐stranded oligonucleotides indicate that Rep75 recognizes the single‐stranded DNA region upstream of the nicking site via non‐covalent interaction and remains covalently linked to the 5′‐phosphate of the downstream fragment after nicking. Besides these expected activities, Rep75 contains a dATP (and ATP) terminal transferase activity at the 3′‐OH extremity of the nicking site, which had not been reported previously for proteins of this type. Rep75, which is the first replication initiator protein characterized in an archaeon, offers an attractive new model for the study of rolling circle replication.

Unexpected role for Helicobacter pylori DNA polymerase I as a source of genetic variability.

Helicobacter pylori, a human pathogen infecting about half of the world population, is characterised by its large intraspecies variability. Its genome plasticity has been invoked as the basis for its high adaptation capacity. Consistent with its small genome, H. pylori possesses only two bona fide DNA polymerases, Pol I and the replicative Pol III, lacking homologues of translesion synthesis DNA polymerases. Bacterial DNA polymerases I are implicated both in normal DNA replication and in DNA repair. We report that H. pylori DNA Pol I 5′- 3′ exonuclease domain is essential for viability, probably through its involvement in DNA replication. We show here that, despite the fact that it also plays crucial roles in DNA repair, Pol I contributes to genomic instability. Indeed, strains defective in the DNA polymerase activity of the protein, although sensitive to genotoxic agents, display reduced mutation frequencies. Conversely, overexpression of Pol I leads to a hypermutator phenotype. Although the purified protein displays an intrinsic fidelity during replication of undamaged DNA, it lacks a proofreading activity, allowing it to efficiently elongate mismatched primers and perform mutagenic translesion synthesis. In agreement with this finding, we show that the spontaneous mutator phenotype of a strain deficient in the removal of oxidised pyrimidines from the genome is in part dependent on the presence of an active DNA Pol I. This study provides evidence for an unexpected role of DNA polymerase I in generating genomic plasticity.

Genetic dissection of Helicobacter pylori AddAB role in homologous recombination

Helicobacter pylori infects the stomach of about half of the worlds human population, frequently causing chronic inflammation at the origin of several gastric pathologies. One of the most remarkable characteristics of the species is its remarkable genomic plasticity in which homologous recombination (HR) plays a critical role. Here, we analyzed the role of the H. pylori homologue of the AddAB recombination protein. Bioinformatics analysis of the proteins unveils the similarities and differences of the H. pylori AddAB complex with respect to the RecBCD and AddAB complexes from Escherichia coli and Bacillus subtilis, respectively. Helicobacter pylori mutants lacking functional addB or/and addA show the same level of sensitivity to DNA-damaging agents such as UV or irradiation and of deficiency in intrachromosomal RecA-dependent HR. Epistasis analyses of both DNA repair and HR phenotypes, using double and triple recombination mutants, demonstrate that, in H. pylori, AddAB and RecOR complexes define two separate presynaptic pathways with little functional overlap. However, neither of these complexes participates in the RecA-dependent process of transformation of these naturally competent bacteria.

The nuclease activities of both the Smr domain and an additional LDLK motif are required for an efficient anti-recombination function of Helicobacter pylori MutS2.

Helicobacter pylori, a human pathogen, is a naturally and constitutively competent bacteria, displaying a high rate of intergenomic recombination. While recombination events are essential for evolution and adaptation of H. pylori to dynamic gastric niches and new hosts, such events should be regulated tightly to maintain genomic integrity. Here, we analyze the role of the nuclease activity of MutS2, a protein that limits recombination during transformation in H. pylori. In previously studied MutS2 proteins, the C‐terminal Smr domain was mapped as the region responsible for its nuclease activity. We report here that deletion of Smr domain does not completely abolish the nuclease activity of HpMutS2. Using bioinformatics analysis and mutagenesis, we identified an additional and novel nuclease motif (LDLK) at the N‐terminus of HpMutS2 unique to Helicobacter and related ε‐proteobacterial species. A single point mutation (D30A) in the LDLK motif and the deletion of Smr domain resulted in ∼ 5–10‐fold loss of DNA cleavage ability of HpMutS2. Interestingly, the mutant forms of HpMutS2 wherein the LDLK motif was mutated or the Smr domain was deleted were unable to complement the hyper‐recombination phenotype of a mutS2− strain, suggesting that both nuclease sites are indispensable for an efficient anti‐recombinase activity of HpMutS2.

[16] pGT5 replication initiator protein Rep75 from Pyrococcus abyssi

Publisher Summary Plasmids are extrachromosomal elements that can replicate autonomously in host cells. They are well known and studied in bacteria and have allowed the development of essential genetic tools in many bacterial species. Plasmids have also been very useful in studying fundamental cellular mechanisms, such as DNA replication. The plasmid pGT5 (3.4 kb), isolated from the euryarchaeon Pyrococcus abyssi , was the first plasmid studied in hyperthermophilic archaea.1 It was completely sequenced and shown to replicate via the rolling circle (RC) mechanism. RC replicons encode a replication initiator protein (usually named Rep) that exhibits a site-specific endonuclease/ligase activity. Several Rep protein encoded by bacterial or eukaryal RC replicons have been studied in vivo and in vitro , and they all share common characteristics. The purified Rep protein of pGT5 (Rep75) exhibits a highly thermophilic and specific nicking-closing (NC) activity on single-stranded oligonucleotides containing the pGT5 double-stranded origin (dso) Sequence. Rep75 exhibits an unusual site-specific nucleotidyl-terminal transferase (NTT) activity, never described before for a Rep protein. The protein Rep75 can be overproduced in Escherichia coli and purified to near homogeneity. The method used, as well as the activity tests, is described in detail here and could be relevant to the study of other hyperthermophilic Rep proteins.

ComB proteins expression levels determine Helicobacter pylori competence capacity

Helicobacter pylori chronically colonises half of the world’s human population and is the main cause of ulcers and gastric cancers. Its prevalence and the increase in antibiotic resistance observed recently reflect the high genetic adaptability of this pathogen. Together with high mutation rates and an efficient DNA recombination system, horizontal gene transfer through natural competence makes of H. pylori one of the most genetically diverse bacteria. We show here that transformation capacity is enhanced in strains defective for recN, extending previous work with other homologous recombination genes. However, inactivation of either mutY or polA has no effect on DNA transformation, suggesting that natural competence can be boosted in H. pylori by the persistence of DNA breaks but not by enhanced mutagenesis. The transformation efficiency of the different DNA repair impaired strains correlates with the number of transforming DNA foci formed on the cell surface and with the expression of comB8 and comB10 competence genes. Overexpression of the comB6-B10 operon is sufficient to increase the transformation capacity of a wild type strain, indicating that the ComB complex, present in the bacterial wall and essential for DNA uptake, can be a limiting factor for transformation efficiency.

Mutations in the nucleotide binding and hydrolysis domains of Helicobacter pylori MutS2 lead to altered biochemical activities and inactivation of its in vivo function

BackgroundHelicobacter pylori MutS2 (HpMutS2), an inhibitor of recombination during transformation is a non-specific nuclease with two catalytic sites, both of which are essential for its anti-recombinase activity. Although HpMutS2 belongs to a highly conserved family of ABC transporter ATPases, the role of its ATP binding and hydrolysis activities remains elusive.ResultsTo explore the putative role of ATP binding and hydrolysis activities of HpMutS2 we specifically generated point mutations in the nucleotide-binding Walker-A (HpMutS2-G338R) and hydrolysis Walker-B (HpMutS2-E413A) domains of the protein. Compared to wild-type protein, HpMutS2-G338R exhibited ~2.5-fold lower affinity for both ATP and ADP while ATP hydrolysis was reduced by ~3-fold. Nucleotide binding efficiencies of HpMutS2-E413A were not significantly altered; however the ATP hydrolysis was reduced by ~10-fold. Although mutations in the Walker-A and Walker-B motifs of HpMutS2 only partially reduced its ability to bind and hydrolyze ATP, we demonstrate that these mutants not only exhibited alterations in the conformation, DNA binding and nuclease activities of the protein but failed to complement the hyper-recombinant phenotype displayed by mutS2-disrupted strain of H. pylori. In addition, we show that the nucleotide cofactor modulates the conformation, DNA binding and nuclease activities of HpMutS2.ConclusionsThese data describe a strong crosstalk between the ATPase, DNA binding, and nuclease activities of HpMutS2. Furthermore these data show that both, ATP binding and hydrolysis activities of HpMutS2 are essential for the in vivo anti-recombinase function of the protein.