Efim I. Golub

Dynamic changes in Rad51 distribution on chromatin during meiosis in male and female vertebrates

Antibodies against human Rad51 protein were used to examine the distribution of Rad51 on meiotic chromatin in mouse spermatocytes and oocytes as well as chicken oocytes during sequential stages of meiosis. We observed the following dynamic changes in distribution of Rad51 during meiosis: (1) in early leptotene nuclei there are multiple apparently randomly distributed, foci that by late leptonema become organized into tracks of foci. (2) These foci persist into zygonema, but most foci are now localized on Rad51-positive axes that correspond to lateral elements of the synaptonemal complex. As homologs synapse foci from homologous axes fuse. The distribution and involvement of Rad51 foci as contact points between homologs suggest that they may be components to early recombination nodules. (3) As pachynema progresses the number of foci drops dramatically; the temporal occurrence (mice) and physical and numerical distribution of foci on axes (chickens) suggest that they may be a component of late recombination nodules. (4) In early pachynema there are numerous Rad51 foci on the single axis of the X (mouse spermatocytes) or the Z (chiken oocytes) chromosomes that neither pair, nor recombine. (5) In late pachynema in mouse spermatocytes, but not oocytes, the Rad51 signal is preferentially enhanced at both ends of all the bivalents. As bivalents in spermatocytes, but not oocytes, begin to desynapse at diplonema they are often held together at these Rad51-positive termini. These observations parallel observations that recombination rates are exceptionally high near chromosome ends in male but not female eutherian mammals. (6) From diakinesis through metaphase I, Rad51 protein is detected as low-intensity fluorescent doublets that localize with CREST-specific antigens (kinetochores), suggesting that Rad51 participates, at least as a structural component of the materials involved, in sister kinetochore cohesiveness. Finally, the changes in Rad51 distribution during meiosis do not appear to be species specific, but intrinsic to the meiotic process.

Human Rad52 protein promotes single-strand DNA annealing followed by branch migration

In the yeast, Saccharomyces cerevisiae, the Rad52 gene is important for both mitotic and meiotic recombination. Homologs of the Rad52 gene have been identified in several eukaryotic organisms, ranging from yeast to man. As reported here, human Rad52 protein binds to both single- and double-stranded DNA; and acting on a pair of single-stranded and partially duplex substrates it promotes annealing of complementary strands of DNA, which is followed by branch migration.

The synaptic activity of HsDmc1, a human recombination protein specific to meiosis.

Human Dmc1 protein, a meiosis-specific homolog of Escherichia coli RecA protein, has previously been shown to promote DNA homologous pairing and strand-exchange reactions that are qualitatively similar to those of RecA protein and Rad51. Human and yeast Rad51 proteins each form a nucleoprotein filament that is very similar to the filament formed by RecA protein. However, recent studies failed to find a similar filament made by Dmc1 but showed instead that this protein forms octameric rings and stacks of rings. These observations stimulated further efforts to elucidate the mechanism by which Dmc1 promotes the recognition of homology. Dmc1, purified to a state in which nuclease and helicase activities were undetectable, promoted homologous pairing and strand exchange as measured by fluorescence resonance energy transfer (FRET). Observations on the intermediates and products, which can be distinguished by FRET assays, provided direct evidence of a three-stranded synaptic intermediate. The effects of helix stability and mismatched base pairs on the recognition of homology revealed further that human Dmc1, like human Rad51, requires the preferential breathing of A⋅T base pairs for recognition of homology. We conclude that Dmc1, like human Rad51 and E. coli RecA protein, promotes homologous pairing and strand exchange by a “synaptic pathway” involving a three-stranded nucleoprotein intermediate, rather than by a “helicase pathway” involving the separation and reannealing of DNA strands.

Ethnic Related Selection for an ADH Class I Variant within East Asia

Background The alcohol dehydrogenases (ADH) are widely studied enzymes and the evolution of the mammalian gene cluster encoding these enzymes is also well studied. Previous studies have shown that the ADH1B*47His allele at one of the seven genes in humans is associated with a decrease in the risk of alcoholism and the core molecular region with this allele has been selected for in some East Asian populations. As the frequency of ADH1B*47His is highest in East Asia, and very low in most of the rest of the world, we have undertaken more detailed investigation in this geographic region. Methodology/Principal Findings Here we report new data on 30 SNPs in the ADH7 and Class I ADH region in samples of 24 populations from China and Laos. These populations cover a wide geographic region and diverse ethnicities. Combined with our previously published East Asian data for these SNPs in 8 populations, we have typed populations from all of the 6 major linguistic phyla (Altaic including Korean-Japanese and inland Altaic, Sino-Tibetan, Hmong-Mien, Austro-Asiatic, Daic, and Austronesian). The ADH1B genotyping data are strongly related to ethnicity. Only some eastern ethnic phyla or subphyla (Korean-Japanese, Han Chinese, Hmong-Mien, Daic, and Austronesian) have a high frequency of ADH1B*47His. ADH1B haplotype data clustered the populations into linguistic subphyla, and divided the subphyla into eastern and western parts. In the Hmong-Mien and Altaic populations, the extended haplotype homozygosity (EHH) and relative EHH (REHH) tests for the ADH1B core were consistent with selection for the haplotype with derived SNP alleles. In the other ethnic phyla, the core showed only a weak signal of selection at best. Conclusions/Significance The selection distribution is more significantly correlated with the frequency of the derived ADH1B regulatory region polymorphism than the derived amino-acid altering allele ADH1B*47His. Thus, the real focus of selection may be the regulatory region. The obvious ethnicity-related distributions of ADH1B diversities suggest the existence of some culture-related selective forces that have acted on the ADH1B region.

Derepression of single-stranded DNA-binding protein genes on plasmids derepressed for conjugation, and complementation of an E. coli ssb- mutation by these genes.

SummaryPlasmid single-stranded DNA-binding protein genes complement the E. coli ssb-1 mutation, and partially restore capacity for DNA synthesis, DNA repair (direct role as well as role in SOS induction) and general recombination. Plasmid mutants derepressed for fertility derived from R1, R64 and R222 show a higher level of complementation compared to the parental repressed plasmids. Derepressed mutants of R222 synthesize more RNA which hybridizes with the ssb gene of the F factor than does the original R222 plasmid. This indicates that plasmid ssb genes are regulated coordinately with fertility genes.

RecA protein-mediated irreversible fixation of an oligodeoxyribonucleotide to specific site in DNA

RecA protein can polymerize on an oligodeoxyribonucleotide to form a filament that finds its homologous sequence in double-stranded DNA. When such an oligonucleotide is linked to psoralen, a photoactivatable DNA intercalator, it irreversibly binds to the homologous site in double stranded DNA as a result of psoralen photoadduct formation at thymidines. The relative efficiency of specific vs. nonspecific binding of an oligonucleotide depended upon the ratio of psoralenated oligonucleotide to total DNA. Na+ ions at concentrations greater than 50 mM eliminated specific binding. Under optimal conditions. the probability of binding of an 80-mer oligonucleotide to a specific site was > 10(5) times greater than that of binding to any single nonspecific site. Under the conditions described, RecA-mediated photoadduction was equally efficient with superhelical and linear double-stranded DNA.