The delivery of a ring-shaped hexameric helicase onto DNA is a fundamental step of DNA replication, conserved in all cellular organisms. We report the biochemical characterization of the bacterial hexameric replicative helicase DnaC of Bacillus subtilis with that of the two replication initiation proteins DnaI and DnaB. We show that DnaI and DnaB interact physically and functionally with the DnaC helicase and mediate its functional delivery onto DNA. Thus, DnaB and DnaI form a pair of helicase loaders, revealing a two-protein strategy for the loading of a replicative helicase. We also present evidence that the DnaC helicase loading mechanism appears to be of the ring-assembly type, proceeding through the recruitment of DnaC monomers and their hexamerization around single-stranded DNA by the coordinated action of DnaI and DnaB.

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 ␤. 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.A major threat to genetic stability is the damaging of DNA by either endogenous or exogenous compounds. This is underscored by the cancer-prone phenotype of human cells defective in DNA repair processes. Exposure of the cellular DNA to reactive oxygen species (ROS), 1 generated either by the normal metabolism of the cell or by chemical and physical exogenous agents, is at the origin of lesions that can have genotoxic or mutagenic consequences. To avoid the effects of ROS and, therefore, to maintain the integrity of their genetic information, organisms have evolved multiple DNA repair mechanisms (1). Because of its capacity to pair with an adenine during replication, 7,8-dihydro-8-oxoguanine (8-oxoG), an oxidized derivative of guanine, is arguably the major mutagenic lesion in DNA. Indeed, in Escherichia coli, the inactivation of the genes involved in the repair of this oxidized base leads to one of the strongest spontaneous mutator phenotypes, characterized by the exclusive increase in G to T transversions. Like for other ROS-induced modifications of DNA, 8-oxoG is mainly repaired by the base excision repair (BER) pathway. This pathway is initiated by the recognition and excision of the oxidized guanine by a DNA glycosylase, OGG1 being the major one in yeast and mammalian cells (2). In human cells, the resulting abasic (apurinic/apyrimidinic (AP)) site can be cleaved by a second enzymatic activity of the hOGG1 polypeptide, namely an AP lyase activity. If such a reaction takes place, the nick produced has a 3Ј-open aldehyde residue that is supposed to be removed by the 3Ј-deoxyribose phosphatase activity of APE1, the major AP endonuclease. However, recent data s...

In addition to their role in DNA repair, recombination events are associated with processes aimed at providing the genetic variability needed for adaptation and evolution of a population. In bacteria, recombination is involved in the appearance of new variants by allowing the incorporation of exogenous DNA or the reshuffling of endogenous sequences. Here we show that HpMutS2, a protein belonging to the MutS2 family in Helicobacter pylori, is not involved in mismatch repair but inhibits homologous and homeologous recombination. Disruption of HpmutS2 leads to an increased efficiency of exogenous DNA incorporation. HpMutS2 has a selective affinity for DNA structures mimicking recombination intermediates with no specificity for homoduplex DNA or mismatches. The purified protein has an ATPase activity stimulated by the same DNA structures. Finally, we show that HpMutS2 inhibits DNA strand exchange reactions in vitro. Thus, MutS2 proteins are candidates for controlling recombination and therefore genetic diversity in bacteria.

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Primosomes are nucleoprotein assemblies designed for the activation of DNA replication forks. Their primary role is to recruit the replicative helicase onto single-stranded DNA. The "replication restart" primosome, defined in Escherichia coli, is involved in the reactivation of arrested replication forks. Binding of the PriA protein to forked DNA triggers its assembly. PriA is conserved in bacteria, but its primosomal partners are not. In Bacillus subtilis, genetic analysis has revealed three primosomal proteins, DnaB, DnaD, and DnaI, that have no obvious homologues in E. coli. Interestingly, they are involved in primosome function both at arrested replication forks and at the chromosomal origin. Our biochemical analysis of the DnaB and DnaD proteins unravels their role in primosome assembly. They are both multimeric and bind individually to DNA. Furthermore, DnaD stimulates DnaB binding activities. DnaD alone and the DnaD/DnaB pair interact specifically with PriA of B. subtilis on several DNA substrates. This suggests that the nucleoprotein assembly is sequential in the PriA, DnaD, DnaB order. The preferred DNA substrate mimics an arrested DNA replication fork with unreplicated lagging strand, structurally identical to a product of recombinational repair of a stalled replication fork.

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 cadmiumtreated 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.

SummaryInitiation and re-initiation of chromosomal DNA replication in bacteria rely on divergent multiprotein assemblies, which direct the functional delivery of the replicative helicase on single-stranded DNA (ssDNA) at specific sites. These two processes are triggered either at the single chromosomal origin oriC or at arrested forks by the conserved DnaA and PriA proteins respectively. In Bacillus subtilis , these two pathways further require the three essential proteins DnaB, DnaD and DnaI, restrictively encoded in Gram positive bacteria of low GC content. We have recently shown that DnaI and DnaB act as a pair of loaders of the DnaC replicative helicase. The role of DnaD appeared more enigmatic. It was previously shown to interact with DnaA and to display weak ssDNA binding activity. Here, we report that purified DnaD can interact physically with PriA and with DnaB. We show that the lethality of the temperature-sensitive dnaD23 mutant can be suppressed by different DnaB point mutants, which were found to be identical to the suppressors of priA null mutants. The DnaD23 protein displays lower ssDNA binding activity than DnaD. Conversely, the DnaB75 protein, the main dnaD23 suppressor, has gained affinity for ssDNA. Finally, we observed that this interplay between DnaD and DnaB is crucial for their concerted interaction with SSBcoated ssDNA, which is the expected substrate for the loading of the replicative helicase in vivo . Altogether, these results highlight the need for both DnaD and DnaB to interact individually and together with ssDNA during the early stages of initiation and re-initiation of chromosomal DNA replication. They also point at a main structural role of DnaD in the multiprotein assemblies built during these two essential processes.

The plasmid pGT5 (3,444 bp) from the hyperthermophilic archaeon Pyrococcus abyssi GE5 has been completely sequenced. Two major open reading frames with a good coding probability are located on the same strand and cover 85% of the total sequence. The larger open reading frame encodes a putative polypeptide which exhibits sequence similarity with Rep proteins of plasmids using the rolling-circle mechanism for replication. Upstream of this open reading frame, we have detected an 11-bp motif identical to the doublestranded origin of several bacterial plasmids that replicate via the rolling-circle mechanism. A putative single-stranded origin exhibits similarities both to bacterial primosome-dependent single-stranded initiation sites and to bacterial primase (dnaG) start sites. A single-stranded form of pGT5 corresponding to the plus strand was detected in cells of P. abyssi. These data indicate that pGT5 replicates via the rolling-circle mechanism and suggest that members of the domain Archaea contain homologs of several bacterial proteins involved in chromosomal DNA replication. Phylogenetic analysis of Rep proteins from rolling-circle replicons suggest that diverse families diverged before the separation of the domains Archaea, Bacteria, and Eucarya.The isolation and characterization of plasmids are prerequisites for the development of genetic studies on new groups of microorganisms. Plasmids also are essential tools for studying in vivo and in vitro mechanisms such as DNA replication, recombination, and repair (28). Bacterial plasmids have been extensively analyzed and used in molecular and genetic work. In contrast, information about plasmids in the domain Archaea (the third domain of life sensu Woese et al. [49]) is much more limited. Several plasmids in extreme halophiles and methanogens have been described, and a few of them were completely sequenced: pHV2 (6.3 kb) from Haloferax volcanii (7), pGRB1 (1.8 kb) from Halobacterium sp. strain GRB and its relatives pGN101 and pHSB1 (18,19,26), and pME2001 (4.4 kb) from Methanobacterium thermoautotrophicum Marburg (4). The minimal replication regions for pHH1 (ϳ150 kb) and pNRC100 (ϳ200 kb) from Halobacterium salinarium (33, 37) and pHK2 (10.5 kb) from Haloferax sp. strain Aa2.2 (24) were defined and sequenced. The plasmid pGRB1 and its relatives, as well as pHK2, encode homologous proteins that exhibit similarities with Rep proteins from rolling-circle (RC) replicons of the X174 group (24, 25). In contrast, pNRC100 and pHV2 encode homologous Rep proteins (encoded by repH) unrelated to those of the RC replicons (33). Some of the haloarchaeal plasmids have been used successfully for the construction of shuttle vectors (23,29,30). The number of plasmids described for thermophilic archaea is restricted compared with those described for halophiles and methanogens. The first extrachromosomal element detected in a member of the order Sulfolobales, pSB12 (15.5 kb) (50), was in fact the genome of a lysogenic virus, Sulfolobus shibatae virus 1 (SSV1) (for a review, see refere...