Biomedical subjects

Gerald R Smith

Publications and source records attributed to Gerald R Smith.

27 records · Page 2Linked to original sources

PubMed2003

Interchangeable parts of the Escherichia coli recombination machinery.

In Escherichia coli, at least two groups of proteins, or "recombination machines," can operate independently on broken DNA to produce a 3'-terminated single-stranded DNA filament coated with RecA protein and ready for synapsis with intact homologous DNA. Recent analyses of mutants lacking one or more of the activities required for presynaptic filament formation by one recombination machine demonstrate that parts of the two normally separate machines can interchange to initiate homologous recombination.

Adenosine Triphosphate↗

PubMed2003

Nonrandom homolog segregation at meiosis I in Schizosaccharomyces pombe mutants lacking recombination.

Physical connection between homologous chromosomes is normally required for their proper segregation to opposite poles at the first meiotic division (MI). This connection is generally provided by the combination of reciprocal recombination and sister-chromatid cohesion. In the absence of meiotic recombination, homologs are predicted to segregate randomly at MI. Here we demonstrate that in rec12 mutants of the fission yeast Schizosaccharomyces pombe, which are devoid of meiosis-induced recombination, homologs segregate to opposite poles at MI 63% of the time. Residual, Rec12-independent recombination appears insufficient to account for the observed nonrandom homolog segregation. Dyad asci are frequently produced by rec12 mutants. More than half of these dyad asci contain two viable homozygous-diploid spores, the products of a single reductional division. This set of phenotypes is shared by other S. pombe mutants that lack meiotic recombination, suggesting that nonrandom MI segregation and dyad formation are a general feature of meiosis in the absence of recombination and are not peculiar to rec12 mutants. Rec8, a meiosis-specific sister-chromatid cohesin, is required for the segregation phenotypes displayed by rec12 mutants. We propose that S. pombe possesses a system independent of recombination that promotes homolog segregation and discuss possible mechanisms.

Base Sequence↗

PubMed2003

Fission yeast Mus81.Eme1 Holliday junction resolvase is required for meiotic crossing over but not for gene conversion.

Most models of homologous recombination invoke cleavage of Holliday junctions to explain crossing over. The Mus81.Eme1 endonuclease from fission yeast and humans cleaves Holliday junctions and other branched DNA structures, leaving its physiological substrate uncertain. We report here that Schizosaccharomyces pombe mus81 mutants have normal or elevated frequencies of gene conversion but 20- to 100-fold reduced frequencies of crossing over. Thus, gene conversion and crossing over can be genetically separated, and Mus81 is required for crossing over, supporting the hypothesis that the fission yeast Mus81.Eme1 protein complex resolves Holliday junctions in meiotic cells.

Crossing Over, Genetic↗

PubMed2002

DNA unwinding step-size of E. coli RecBCD helicase determined from single turnover chemical quenched-flow kinetic studies.

The mechanism by which Escherichia coli RecBCD DNA helicase unwinds duplex DNA was examined in vitro using pre-steady-state chemical quenched-flow kinetic methods. Single turnover DNA unwinding experiments were performed by addition of ATP to RecBCD that was pre-bound to a series of DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. In each case, the time-course for formation of completely unwound DNA displayed a distinct lag phase that increased with duplex length, reflecting the transient formation of partially unwound DNA intermediates during unwinding catalyzed by RecBCD. Quantitative analysis of five independent sets of DNA unwinding time courses indicates that RecBCD unwinds duplex DNA in discrete steps, with an average unwinding "step-size", m=3.9(+/-1.3)bp step(-1), with an average unwinding rate of k(U)=196(+/-77)steps s(-1) (mk(U)=790(+/-23)bps(-1)) at 25.0 degrees C (10mM MgCl(2), 30 mM NaCl (pH 7.0), 5% (v/v) glycerol). However, additional steps, not linked directly to DNA unwinding are also detected. This kinetic DNA unwinding step-size is similar to that determined for the E.coli UvrD helicase, suggesting that these two SF1 superfamily helicases may share similar mechanisms of DNA unwinding.

Adenosine Triphosphate↗

PubMed2002

Meiotic recombination remote from prominent DNA break sites in S. pombe.

DNA breakage is intimately associated with meiotic recombination in the fission yeast Schizosaccharomyces pombe. Sites of prominent DNA breakage were found approximately 25 to approximately 200 kb apart in the genomic regions surveyed. We examined in detail a 501 kb region of chromosome I and found six sites, or tight clusters of sites, at which approximately 2%-11% of the DNA accumulated breaks in a rad50S mutant. In contrast to the discrete, widely spaced distribution of prominent break sites, recombination in this region was more uniformly distributed (0.7-1.6 cM/10 kb) whether the genetic interval tested contained no, one, or more such sites. We infer that although recombination depends upon DNA breakage, recombination often occurs remote from these sites (tens of kilobases away); we discuss mechanisms by which this may occur.

Chromosome Breakage↗

PubMed2002

Meiotic DNA breaks at the S. pombe recombination hot spot M26.

The ade6-M26 allele of Schizosaccharomyces pombe creates a well-defined meiotic recombination hot spot that requires a specific sequence, 5'-ATGACGT-3', and the Atf1*Pcr1 transcription factor for activity. We find that M26 stimulates the formation of meiosis-specific double-strand DNA breaks at multiple sites surrounding M26. Like hot spot activity, breakage requires the M26 heptamer, Pcr1, and the general recombination factor Rec12. When the M26 heptamer is moved to new positions within ade6, new break sites are observed spanning approximately 0.5-2 kb around the moved heptamer. Break frequency is strongly correlated with recombination frequency for these alleles. The occurrence of breaks at M26 suggests mechanistic similarities to hot spots in the distantly related yeast Saccharomyces cerevisiae.

Activating Transcription Factor 1↗

PubMed2002

A 160-bp palindrome is a Rad50.Rad32-dependent mitotic recombination hotspot in Schizosaccharomyces pombe.

Palindromic sequences can form hairpin and cruciform structures that pose a threat to genome integrity. We found that a 160-bp palindrome (an inverted repeat of 80 bp) conferred a mitotic recombination hotspot relative to a control nonpalindromic sequence when inserted into the ade6 gene of Schizosaccharomyces pombe. The hotspot activity of the palindrome, but not the basal level of recombination, was abolished by a rad50 deletion, by a rad50S "separation of function" mutation, or by a rad32-D25A mutation in the nuclease domain of the Rad32 protein, an Mre11 homolog. We propose that upon extrusion of the palindrome the Rad50.Rad32 nuclease complex recognizes and cleaves the secondary structure thus formed and generates a recombinogenic break in the DNA.

DNA-Binding Proteins↗

PubMed2002

A domain of RecC required for assembly of the regulatory RecD subunit into the Escherichia coli RecBCD holoenzyme.

The heterotrimeric RecBCD enzyme of Escherichia coli is required for the major pathway of double-strand DNA break repair and genetic exchange. Assembled as a heterotrimer, the enzyme has potent nuclease and helicase activity. Analysis of recC nonsense and deletion mutations revealed that the C terminus of RecC is required for assembly of the RecD subunit into RecBCD holoenzyme but not for recombination proficiency; the phenotype of these mutations mimics that of recD deletion mutations. Partial proteolysis of purified RecC polypeptide yielded a C-terminal fragment that corresponds to the RecD-interaction domain. RecD is essential for nuclease activity, regulation by the recombination hotspot Chi, and high affinity for DNA ends. The RecC-RecD interface thus appears critical for the regulation of RecBCD enzyme via the assembly and, we propose, disassembly or conformational change of the RecD subunit.

DNA, Bacterial↗

PubMed2002

Maximal power tests for detecting defects in meiotic recombination.

We have determined the marker separations (genetic distances) that maximize the probability, or power, of detecting meiotic recombination deficiency when only a limited number of meiotic progeny can be assayed. We find that the optimal marker separation is as large as 30-100 cM in many cases. Provided the appropriate marker separation is used, small reductions in recombination potential (as little as 50%) can be detected by assaying a single interval in as few as 100 progeny. If recombination is uniformly altered across the genomic region of interest, the same sensitivity can be obtained by assaying multiple independent intervals in correspondingly fewer progeny. A reduction or abolition of crossover interference, with or without a reduction of recombination proficiency, can be detected with similar sensitivity. We present a set of graphs that display the optimal marker separation and the number of meiotic progeny that must be assayed to detect a given recombination deficiency in the presence of various levels of crossover interference. These results will aid the optimal design of experiments to detect meiotic recombination deficiency in any organism.

Meiosis↗