Plasmids carrying gene pairs encoding type II DNA restriction endonucleases and their cognate modification enzymes were shown to have increased stability in Escherichia coli. The descendants of cells that had lost these genes appeared unable to modify a sufficient number of recognition sites in their chromosomes to protect them from lethal attack by the remaining restriction enzyme molecules. The capacity of these genes to act as a selfish symbiont is likely to have contributed to the evolution of restriction-modification gene pairs.
Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability. The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex. In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the EcoRI restriction modification gene complex carried by a temperature-sensitive plasmid in an Escherichia coli strain that is wild type with respect to DNA repair. A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability. Many cells formed long filaments, some of which were multinucleated and others anucleated. In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated. Growth inhibition was also more severe in recA, ruvAB, ruvC, recG, and recN mutants. The cells induced the SOS response in a RecBC-dependent manner. These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes. These and previous results have led us to hypothesize that the RecBCD/ Chi/RecA system serves to destroy restricted "nonself" DNA and repair restricted "self" DNA.A type II restriction enzyme, such as R.EcoRI, will make a double-stranded break at a specific sequence on DNA (49). A cognate modification enzyme (M.EcoRI) can methylate the same sequence and protect it from restriction cleavage. The genes involved are tightly linked and form a type II restrictionmodification (RM) gene complex. Type II RM gene complexes will attack unmodified foreign DNA such as bacteriophage DNA but not the modified DNA of the cells where they reside. They have been considered to function as bacterial tools against invasion by foreign DNA.We found that elimination of type II RM gene complexes from bacterial cells by a competing genetic element inhibits cell growth (43,44). Our experiments suggested the following course of events after a cell has lost a type II RM gene complex. In the descendants of the cell that have lost the type II RM gene complex, the number of molecules of the modification enzyme will decrease with each cell division. Eventually, the capacity of the enzyme to modify the many sites needed to protect the newly replicated chromosomes from the remaining pool of restriction enzyme will become inadequate. Chromosomal DNA will then be cleaved at the unmodified sites, and the cells will be killed. This is reminiscent of postsegregational cell killing mechanisms, which have been shown to contribute to the stable maintenance of plasmids (11,12,21). Indeed, linkage of several type II RM gene complexes stabilizes plasmids (29,32,43...
Restriction-modification (RM) systems are believed to have evolved to protect cells from foreign DNA. However, this hypothesis may not be sufficient to explain the diversity and specificity in sequence recognition, as well as other properties, of these systems. We report that the EcoRI restriction endonuclease-modification methylase (rm) gene pair stabilizes plasmids that carry it and that this stabilization is blocked by an RM of the same sequence specificity (EcoRI or its isoschizomer, Rsr I) but not by an RM of a different specificity (PaeR7I) on another plasmid. The PaeR7I rm likewise stabilizes plasmids, unless an rm gene pair with identical sequence specificity is present. Our analysis supports the following model for stabilization and incompatibility: the descendants of cells that have lost an rm gene pair expose the recognition sites in their chromosomes to lethal attack by any remaining restriction enzymes unless modification by another RM system of the same specificity protects these sites. Competition for specific sequences among these selfish genes may have generated the great diversity and specificity in sequence recognition among RM systems. Such altruistic suicide strategies, similar to those found in virusinfected cells, may have allowed selfish RM systems to spread by effectively competing with other selfish genes.A type II restriction endonuclease makes a double-strand break within or near a specific recognition sequence in duplex DNA. A cognate modification enzyme methylates the recognition sequence to protect it from the cleavage (1, 2). It is widely accepted that the evolution and maintenance of restriction-modification (RM) systems have been driven by the protection from foreign DNA that they afford to cells. The RM systems do protect cells from infection with some viruses by cleaving their DNA (for example, see ref.3) and are likely to be responsible both for the evolution of antirestriction mechanisms and for the paucity of some restriction sites in certain viruses and plasmids (4).Recent experimental and theoretical analyses (5, 6), however, seem to us to bring into question the efficacy of virusmediated selection for RM systems. Defense by RM systems is short-lived because invading viral DNA will occasionally escape restriction and will become modified, thus affording protection from restriction to itself and its descendants (5, 6). Bacteria will more likely develop other, longer-lasting means of resistance to viruses, such as alterations in the receptor required for infection (5, 6). Although RM systems can provide bacteria with advantage when they are invading new habitats full of phages, it is not clear whether such colonization selection is realistic under natural conditions (5, 6).It is also unclear whether the above "cellular defense" hypothesis can account for the following properties of type II RM systems (1, 2). (i) Their individual high specificity and collective wide diversity in the sequence recognition. (ii) The tight linkage of cognate restriction and modification ...
The Drosophila Dmblm locus is a homolog of the human Bloom syndrome gene, which encodes a helicase of the RECQ family. We show that Dmblm is identical to mus309, a locus originally identified in a mutagen-sensitivity screen. One mus309 allele, which carries a stop codon between two of the helicase motifs, causes partial male sterility and complete female sterility. Mutant males produce an excess of XY sperm and nullo sperm, consistent with a high frequency of nondisjunction and/or chromosome loss. These phenotypes of mus309 suggest that Dmblm functions in DNA double-strand break repair. The mutant Dmblm phenotypes were partially rescued by an extra copy of the DNA repair gene Ku70, indicating that the two genes functionally interact in vivo.
Background: x sequence (5 0 GCTGGTGG) of Escherichia coli was first identified as a site that increased the plaque size of bacteriophage l. Subsequent studies showed that this site is responsible for both the attenuation of RecBCD exonuclease activity and the promotion of RecA, RecBCD-mediated recombination. It is known that bacteriophage l containing the x site makes very small plaques on a recC* (recC1004) mutant because x is not recognized by the RecBC*D mutant enzyme.
DNA damage alone or DNA replication fork arrest at damaged sites may induce DNA double-strand breaks and initiate homologous recombination. This event can result in a crossover with a homologous chromosome, causing loss of heterozygosity along the chromosome. It is known that Srs2 acts as an antirecombinase at the replication fork: it is recruited by the SUMO (a small ubiquitin-related modifier)-conjugated DNA-polymerase sliding clamp (PCNA) and interferes with Rad51/Rad52-mediated homologous recombination. Here, we report that Srs2 promotes another type of homologous recombination that produces noncrossover products only, in collaboration with PCNA and Rad51. Srs2 proteins lacking the Rad51-binding domain, PCNA-SUMO-binding motifs, or ATP hydrolysis-dependent DNA helicase activity reduce this noncrossover recombination. However, the removal of either the Rad51-binding domain or the PCNA-binding motif strongly increases crossovers. Srs2 gene mutations are epistatic to mutations in the PCNA modification-related genes encoding PCNA, Siz1 (a SUMO ligase) and Rad6 (a ubiquitin-conjugating protein). Knocking out RAD51 blocked this recombination but enhanced nonhomologous end-joining. We hypothesize that, during DNA double-strand break repair, Srs2 mediates collaboration between the Rad51 nucleofilament and PCNA-SUMO and directs the heteroduplex intermediate to DNA synthesis in a moving bubble. This Rad51/Rad52/Srs2/PCNAmediated noncrossover pathway avoids both interchromosomal crossover and imprecise end-joining, two potential paths leading to loss of heterozygosity, and contributes to genome maintenance and human health.utations in the DNA helicase Srs2 gene cause a hyperrecombination phenotype (1) and increase mitotic crossovers (2). These findings suggest that Srs2 negatively regulates somatic homologous recombination, and thus Srs2 is regarded as an antirecombinase. The Srs2 DNA helicase has a recombinase Rad51-binding motif in its C-terminal region (3, 4), and in vitro analyses have demonstrated that it disrupts Rad51 nucleofilaments formed on single-stranded DNA and inhibits heteroduplex formation mediated by Rad51 recombinase (4, 5). In addition, synthetic heteroduplexes with a D-loop with Rad51 nucleofilaments are efficiently dissociated by Srs2 (6). These biochemical results appear to explain the negative regulation of heteroduplex formation by Srs2 during homologous recombination. Other motifs around the C-terminal tip allow the Srs2 helicase to bind to SUMO-conjugated DNA-polymerase sliding clamp (PCNA-SUMO) (3, 7). The interaction between Srs2 and PCNA-SUMO is essential to prevent the Rad51/Rad52-mediated sister-chromatid exchanges that occur in DNA replication forks stalled at DNA lesions, to channel to the translesion DNA synthesis initiated by Rad6/Rad18-ubiquitinated PCNA (3,8). This postreplication repair is induced by base-modification types of DNA lesions, caused by DNA scission reagents such as UV, 4NQO, and MMS.Radiation and reactive oxygen species cause DNA doublestrand breaks, which are repaire...
Whether or not homologous interaction of two DNA molecules results in crossing-over of the flanking sequences is an important decision in view of genome organization. Several homologous recombination models, including the double-strand break repair models, explain this decision as choice between two alternative modes of resolution of Hollidaytype intermediates. We have demonstrated that a doublestrand gap can be repaired through gene conversion copying a homologous duplex, as predicted by the double-strand break repair models, in the RecE pathway of Escherichia coli. This gap repair is often accompanied by crossing-over of the flanking sequences. Mutations in ruvC and recG, whose products interact with Holliday structures in vitro, do not block doublestrand gap repair or its association with flanking crossing-over. However, two mutations in the recJ gene, which encodes a single-strand 5' -+3' exonuclease, severely decrease association of flanking crossing-over. Two mutations in the recQ gene, which encodes a helicase, moderately decrease association of flanking crossing-over by themselves and suppress the severe effect of a red mutation. Similar relationships of recJ and recQ mutations are observed in cell survival after ultraviolet light irradiation, vray irradiation, and H202 treatment. We discuss how cooperation of the recQ gene product and the red gene product brings about double-strand break repair accompanied by flanking crossing-over. We also discuss how this reaction is related to repair of chromosome damages.Homologous interaction between two DNA segments may or may not result in crossing-over of the flanking sequences (Fig. 1A). Since such crossing-over could cause gross changes in genome organization, such as deletion, inversion, and translocation, the choice between crossing-over and non-crossing-over may be an important decision. The Holliday model and its descendant models of homologous recombination explain this decision as a choice between two alternative modes of resolution of Holliday structure or related intermediate structures (1-3). In fact, some Holliday resolvases recognize specific sequences at the Holliday joint and promote only one mode of resolution (4). But it is not known whether this reflects such a choice in vivo.The double-strand break repair models (Fig. 1A) (2, 5) propose that homologous recombination is initiated by a double-stranded break on one of the two DNA duplexes. The double-stranded break is repaired by copying a homologous duplex. In the models the intermediates are resolved with or without crossing-over of the flanking sequences (Fig. 1A). We have demonstrated in Escherichia coli the double-strand break repair reaction predicted by these models (5-8). In this work we show that this repair is frequently accompanied by crossing-over. We looked for genetic determinants affecting the crossing-over association with particular interest in two proteins, RuvC and RecG, recently shown to catalyze processing of Holliday structure in vitro (9-11).
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