We have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae and contributes to the shortened lifespan of dna2 mutants. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We show directly that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of doublestrand breaks near the RFB. Both pausing and breakage are elevated in the early aging, hypomorphic dna2-2 helicase mutant. Deletion of FOB1, encoding the fork barrier protein, suppresses the elevated pausing and DSB formation, and represses initiation at rDNA ARSs. The dna2-2 mutation is synthetically lethal with ⌬rrm3, encoding another DNA helicase involved in rDNA replication. It does not appear to be the case that the rDNA is the only determinant of genome stability during the yeast lifespan however since strains carrying deletion of all chromosomal rDNA but with all rDNA supplied on a plasmid, have decreased rather than increased lifespan. We conclude that the replication-associated defects that we can measure in the rDNA are symbolic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.Replication fork stress has been implicated as a major cause of genome instability in bacteria and yeast. In Escherichia coli, replication forks initiated at the origins frequently stall because of mutations in replication proteins, template blocks, or pauses at natural replication terminator sites. A common intermediate in restoring replication forks after stalling is a double-strand break (DSB), 1 which is thought to lead to recombination, producing genomic instability.Evidence that replication forks pause in Saccharomyces cerevisiae is also convincing (1, 2). In the presence of the replication inhibitor HU, forks stall and give rise to single-stranded regions at the forks. In the absence of checkpoint function, the stalled forks are converted to regressed forks, a Holliday-like structure arising by branch migration and reannealing of nascent DNA strands (3). Primase mutants also show high levels of stalled and regressed forks (1). There is also a naturally occurring replication fork barrier (RFB) within the rDNA (ribosomal DNA) repeats, and the structure of forks paused at the RFB has been characterized (4 -6). Finally, recent evidence suggests that the yeast ATR homolog, Mec1, protects replication forks from collapsing and giving rise to DSBs in replication slow zones throughout the chromosome (7). Evidence is also accumulating that replication fork failure leads to recombinogenic structures that result in gross chromosomal rearrangements (7-10). Such events may lead to the well documented genomic instability observed in DNA replication and checkpoint mutants (8, 9). We have proposed that replication fork stallin...

SummaryA strain of Escherichia coli in which both the seqA and mukB genes were inactivated displayed partial suppressions of their individual phenotypes. Temperature sensitivity, anucleate cell production and poor nucleoid folding seen in the mukB strain were suppressed by the seqA null mutation, whereas ®la-mentation, asymmetric septation and compact folding of the nucleoids observed in the seqA strain were suppressed by inactivation of the mukB gene function. However, the asynchronous initiation of chromosome replication in the seqA strain was not reversed in the mukBseqA double mutant. Membrane-associated nucleoids were isolated from the wild-type, mukB, seqA and mukBseqA strains and their sedimentation rates were compared under identical conditions. Whereas the mukB mutation caused unfolding of the nucleoid, the seqA mutation led to a more compact packaging of the chromosome. The mukBseqA double mutant regained the wild-type nucleoid organization as revealed from its rate of sedimentation. Microscopic appearances of the nucleoids were consistent with the sedimentation pro®les. The mukB mutant was oversensitive to novobiocin and this susceptibility was suppressed in the mukBseqA strain, suggesting possible roles of MukB and SeqA in maintaining chromosome topology. The mutual phenotypic suppression of mukB and seqA alleles thus suggests that these genes have opposing in¯u-ences on the organization of the bacterial nucleoid.

Bacterial infections can be aggravated by antibiotic treatment that induces SOS response and vesiculation. This leads to a hypothesis concerning association of SOS with vesiculation. To test it, we conducted multiple analyses of outer membrane vesicles (OMVs) produced from thePseudomonas aeruginosawild type in which SOS is induced by ciprofloxacin and from the LexA noncleavable (lexAN) strain in which SOS is repressed. The levels of OMV proteins, lipids, and cytotoxicity increased for both the treated strains, demonstrating vesiculation stimulation by the antibiotic treatment. However, the further increase was suppressed in thelexANstrains, suggesting the SOS involvement. Obviously, the stimulated vesiculation is attributed by both SOS-related and unrelated factors. OMV subproteomic analysis was performed to examine these factors, which reflected the OMV-mediated cytotoxicity and the physiology of the vesiculating cells under treatment and SOS. Thus, SOS plays a role in the vesiculation stimulation that contributes to cytotoxicity.

Surprisingly, the contribution of defects in DNA replication to the determination of yeast life span has never been directly investigated. We show that a replicative yeast helicase/nuclease, encoded by DNA2 and a member of the same helicase subfamily as the RecQ helicases, is required for normal life span. All of the phenotypes of old wild-type cells, for example, extended cell cycle time, age-related transcriptional silencing defects, and nucleolar reorganization, occur after fewer generations in dna2 mutants than in the wild type. In addition, the life span of dna2 mutants is extended by expression of an additional copy of SIR2 or by deletion of FOB1, which also increase wild-type life span. The ribosomal DNA locus and the nucleolus seem to be particularly sensitive to defects in dna2 mutants, although in dna2 mutants extrachromosomal ribosomal circles do not accumulate during the aging of a mother cell. Several other replication mutations, such as rad27⌬, encoding the FEN-1 nuclease involved in several aspects of genomic stability, also show premature aging. We propose that replication fork failure due to spontaneous, endogenous DNA damage and attendant genomic instability may contribute to replicative senescence. This may imply that the genomic instability, segmental premature aging symptoms, and cancer predisposition associated with the human RecQ helicase diseases, such as Werner, Bloom, and Rothmund-Thomson syndromes, are also related to replicative stress.Saccharomyces cerevisiae provides a promising model system for the identification and study of genetic pathways involved in determining longevity in more complex organisms (25). In yeast there is a spiral form of aging in which mother cells in each division increase in age by one generation, while each new bud is produced at age zero (33, 67). The total life span of yeast is counted as the number of times the mother cell is able to bud. This form of aging resembles mammalian cell replicative senescence (28), the finite number of divisions of a cell in culture, with two differences. Yeast cell division is asymmetric rather than symmetric with respect to age, and yeast cells lyse at the end of the life span rather than undergoing some form of differentiation. We have been interested in using yeast to study the mechanism underlying certain human premature aging and cancer susceptibility syndromes.The yeast SGS1 gene encodes a homolog of the WRN, BLM, and RecQ4 helicases, members of the RecQ family. Mutations in these human genes cause disorders of premature aging and/or cancer susceptibility (16,17,45,98). The helicase encoded by SGS1 is thought to be involved in the down-regulation of homologous recombination in the ribosomal DNA (rDNA) repeats. sgs1⌬ mutants show a severely reduced average life span and are therefore of interest in understanding the molecular mechanisms of human diseases affecting homologous genes (80). Based on the phenotypes of aging that seemed to occur early in sgs1⌬ mutants as well as on other findings, it was proposed early on that...

We have used ethidium bromide titration for direct measurement of the changes in the negative supercoiling of Escherichia coli chromosome caused by mutations inactivating the cell cycle functions mukB and seqA. The amounts of the intercalative agent required to relax the supercoiled chromosome in mukB and seqA mutants were lower and higher, respectively, than for the wild-type parent, confirming that these cell cycle genes modulate the topology of the E. coli chromosome. Plasmid superhelicity measured in these mutant strains showed similar effects albeit of reduced magnitude. As the effects of mukB and seqA mutations were not restricted to the chromosome alone, MukB and SeqA proteins possibly interact with factors involved in the maintenance of intracellular DNA topology. To our knowledge, this is the first direct demonstration of the influence of mukB and seqA genes on the superhelicity of the E. coli chromosome.