Replication arrest leads to the occurrence of DNA double-stranded breaks (DSB). We studied the mechanism of DSB formation by direct measure of the amount of in vivo linear DNA in Escherichia coli cells that lack the RecBCD recombination complex and by genetic means. The RuvABC proteins, which catalyze migration and cleavage of Holliday junctions, are responsible for the occurrence of DSBs at arrested replication forks. In cells proficient for RecBC, RuvAB is uncoupled from RuvC and DSBs may be prevented. This may be explained if a Holliday junction forms upon replication fork arrest, by annealing of the two nascent strands. RecBCD may act on the double-stranded tail prior to the cleavage of the RuvAB-bound junction by RuvC to rescue the blocked replication fork without breakage.

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...

Bacilus subtilis cells selected for their resistance to rhodamine 6G demonstrated a multidrug-resistance (MDR) phenotype resembling that of mammalian MDR cells. Like MDR in mammalian cells, MDR in bacteria was mediated by the efflux of the drugs from the cells. The bacterial multidrug efflux system transported similar drugs and was sensitive to similar inhibitors as the mammalian multidrug transporter, P-glycoprotein. The gene coding for the bacterial multidrug transporter, like the P-glycoprotein gene in mammalian MDR cells, was amplified in the resistant bacteria. On the other hand, the bacterial multidrug transporter showed no sequence similarity to P-glycoprotein but exhibited an obvious homology to tetracycline efflux pumps and carbohydrate-ion symporters. These results show that the transport of structurally unrelated molecules can be mediated by members of different families of membrane transporters.Mammalian cells selected in culture for resistance to various lipophilic cytotoxic drugs often demonstrate resistance not only to the selective agent but also to a large group of apparently unrelated toxic compounds. This phenomenon, called multidrug resistance (MDR), is based on active efflux of drugs from the cells, performed by a membrane ATPase pump, P-glycoprotein (reviews in refs. 1 and 2). In MDR cells, the mdrl gene coding for this protein is frequently amplified (1). Overexpression of the P-glycoprotein gene is believed to be responsible for clinical drug resistance in many tumors.The most intriguing question in the area of MDR is the mechanism of extremely broad chemical specificity of P-glycoprotein. Its substrates have almost nothing in common, except that most of them bear positive electric charge and all of them are moderately hydrophobic. Some substances, such as reserpine and verapamil, are potent inhibitors of P-glycoprotein activity, apparently by a competition mechanism (3, 4). P-glycoprotein belongs to a large family of membrane ATPase pumps both of eukaryotic and of prokaryotic origin (2). It is, however, the only member of this family with a proven ability to transport multiple drugs. Even close homologues ofP-glycoprotein-the mammalian mdr2 gene product (5) and the yeast STE6 protein (6)-do not share this property. Here we describe MDR in Gram-positive bacteria and characterize the gene of a bacterial multidrug transporter.s MATERIALS AND METHODS Bacteria and Plasmids. Bacillus subtilis BD170 (trpC2, thr-S) and BD224 (trpC2, thr-S, recE4), Escherichia coli JM103, B. subtilis plasmids pCB20 (7) and pUB110, E. coli plasmids pUC19 and pBluescript KS(+) (Stratagene), and shuttle plasmid pMK3 (8) were used in this work.Selection of Resistant Bacteria and Sensitivity Assay. BD170 bacteria were selected with rhodamine 6G in the liquid antibiotic medium 3 (Difco) supplemented with 0.4% glucose.Bacteria were grown at 370C with the drug at 0.5, 1, 2, 3, and 4,g/ml, consecutively. The cultures were diluted 1:100 with fresh drug-containing medium every 1-2 days. The drug concentration was in...

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...

Replication fork arrest is a source of genome rearrangements, and the recombinogenic properties of blocked forks are likely to depend on the cause of blockage. Here we study the fate of replication forks blocked at natural replication arrest sites. For this purpose, Escherichia coli replication terminator sequences Ter were placed at ectopic positions on the bacterial chromosome. The resulting strain requires recombinational repair for viability, but replication forks blocked at Ter are not broken. Linear DNA molecules are formed upon arrival of a second round of replication forks that copy the DNA strands of the ®rst blocked forks to the end. A model that accounts for the requirement for homologous recombination for viability in spite of the lack of chromosome breakage is proposed. This work shows that natural and accidental replication arrests sites are processed differently.