Impairment of replication fork progression is a serious threat to living organisms and a potential source of genome instability. Studies in prokaryotes have provided evidence that inactivated replication forks can restart by the reassembly of the replication machinery. Several strategies for the processing of inactivated replication forks before replisome reassembly have been described. Most of these require the action of recombination proteins, with different proteins being implicated, depending on the cause of fork arrest. The action of recombination proteins at blocked forks is not necessarily accompanied by a strand-exchange reaction and may prevent rather than repair fork breakage. These various restart pathways may reflect different structures at stalled forks. We review here the different strategies of fork processing elicited by different kinds of replication impairments in prokaryotes and the variety of roles played by recombination proteins in these processes.W ork from several laboratories has established that in bacteria, a recombination event can lead to the establishment of a unidirectional replication fork (reviewed in refs. 1-3). These observations extend the concept of a direct recombination-replication connection, originally proposed Ϸ30 years ago from studies of bacteriophage T4 and replication. Furthermore, the existence of recombination-dependent replication in yeast suggests that this connection may be a widely distributed phenomenon (4, 5).Considering the tight control of replication initiation at chromosomal origins, the assembly of a complete replisome at recombination intermediates, independently of time and place, is paradoxical. One of the raisons d'être of this potentially dangerous process was revealed by the finding that recombination intermediates form at inactivated replication forks and are used for replication restart. However, studies of the replicationrecombination connection in Escherichia coli have indicated that recombination proteins do not necessarily catalyze strand exchange at blocked forks and rather act in a variety of reactions that depend on the origin of the arrest. We review here the diversity of fates of inactivated replication forks in bacteria, as well as our present knowledge of the roles played by recombination proteins during replication restart. DNA Double-Strand End Repair in BacteriaAlmost 30 years ago, Higgins et al. (6) proposed that blocked replication forks could be isomerized into a four-way Holliday junction (HJ) with a DNA double-strand end, which could permit DNA repair and then continuation of replication (Fig. 1, step A). The test of the model, performed by treatment of mammalian cells with a DNA-damaging agent, was inconclusive (7). However, more recent studies in E. coli suggest that such replication fork reversal plays a crucial role in replication restart in bacteria (8,9).A key aspect of the replication fork reversal model is that it generates a DNA double-strand end at blocked forks without chromosome breakage. In E. coli, the enzyme th...

DNA synthesis is an accurate and very processive phenomenon; nevertheless, replication fork progression on chromosomes can be impeded by DNA lesions, DNA secondary structures, or DNA-bound proteins. Elements interfering with the progression of replication forks have been reported to induce rearrangements and͞or render homologous recombination essential for viability, in all organisms from bacteria to human. Arrested replication forks may be the target of nucleases, thereby providing a substrate for doublestrand break repair enzyme. For example in bacteria, direct fork breakage was proposed to occur at replication forks blocked by a bona fide replication terminator sequence, a specific site that arrests bacterial chromosome replication. Alternatively, an arrested replication fork may be transformed into a recombination substrate by reversal of the forked structures. In reversed forks, the last duplicated portions of the template strands reanneal, allowing the newly synthesized strands to pair. In bacteria, this reaction was proposed to occur in replication mutants, in which fork arrest is caused by a defect in a replication protein, and in UV irradiated cells. Recent studies suggest that it may also occur in eukaryote organisms. We will review here observations that link replication hindrance with DNA rearrangements and the possible underlying molecular processes. Large genome rearrangements, such as duplications, deletions, translocations and insertions, are mainly catalyzed by three classes of molecular processes that differ by length of homology of the joined sequences. Recombination events formed by joining of homologous sequences are mediated by specific enzymes. The key enzyme, RecA in prokaryotes and RecAhomologues in eukaryotes, catalyzes the strand exchange reaction and is highly conserved from bacteria to human (1, 2). A second type of recombination, called illegitimate, is characterized by the joining sequences whose length is below the minimal length required for recognition of homology by RecA (Minimal Efficient Processing Segment, or MEPS). In prokaryotes, illegitimate recombination can result from simple ligation of unrelated sequences (reviewed in ref.3). In eukaryotes, this process is promoted by a battery of specialized enzymes and is called nonhomologous end-joining (NHEJ; reviewed in refs. 4 and 5). In addition, recombination between tandemly repeated sequences forms a distinct class of events that may be catalyzed by several specific pathways (reviewed in ref. 6). All classes of DNA rearrangements are important in human health, as they may cause cancers (reviewed in ref. 7) or hereditary disorders (reviewed in refs. 8 and 9) and are important in evolution (ref. 10; reviewed in ref. 11). All classes of rearrangements can result from the formation and repair of DNA double-strand breaks (DSBs) and have been shown to occur at an increased frequency in DNA regions difficult to replicate or when DNA replication is affected by a mutation. The correlation between replication hindrance and rearrangem...

In an attempt to explore the microbial content of functionally critical niches of the mouse gastrointestinal tract, we targeted molecular microbial diagnostics of the crypts that contain the intestinal stem cells, which account for epithelial regeneration. As current evidence indicates, the gut microbiota affects epithelial regeneration; bacteria that are likely to primarily participate in this essential step of the gut, microbiota cross talk, have been identified. We show in this article that only the cecal and colonic crypts harbor resident microbiota in the mouse and that regardless of the line and breeding origin of these mice, this bacterial population is unexpectedly dominated by aerobic genera. Interestingly, this microbiota resembles the restricted microbiota found in the midgut of invertebrates; thus, the presence of our so-called “crypt-specific core microbiota” (CSCM) in the mouse colon potentially reflects a coevolutionary process under selective conditions that can now be addressed. We suggest that CSCM could play both a protective and a homeostatic role within the colon. This article is setting the bases for such studies, particularly by providing a bona fide—and essentially cultivable—crypt microbiota of reference.

Numerous studies have shown that resistance to oxidative stress is crucial to stay healthy and to reduce the adverse effects of aging. Accordingly, nutritional interventions using antioxidant food-grade compounds or food products are currently an interesting option to help improve health and quality of life in the elderly. Live lactic acid bacteria (LAB) administered in food, such as probiotics, may be good antioxidant candidates. Nevertheless, information about LAB-induced oxidative stress protection is scarce. To identify and characterize new potential antioxidant probiotic strains, we have developed a new functional screening method using the nematode Caenorhabditis elegans as host. C. elegans were fed on different LAB strains (78 in total) and nematode viability was assessed after oxidative stress (3 mM and 5 mM H2O2). One strain, identified as Lactobacillus rhamnosus CNCM I-3690, protected worms by increasing their viability by 30% and, also, increased average worm lifespan by 20%. Moreover, transcriptomic analysis of C. elegans fed with this strain showed that increased lifespan is correlated with differential expression of the DAF-16/insulin-like pathway, which is highly conserved in humans. This strain also had a clear anti-inflammatory profile when co-cultured with HT-29 cells, stimulated by pro-inflammatory cytokines, and co-culture systems with HT-29 cells and DC in the presence of LPS. Finally, this Lactobacillus strain reduced inflammation in a murine model of colitis. This work suggests that C. elegans is a fast, predictive and convenient screening tool to identify new potential antioxidant probiotic strains for subsequent use in humans.