Gene Expression And Human Genetics

Diabetes, Digestive, Kidney Diseases

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Abstract

Summary of work: In order to dissect the biochemical steps involved in genetic recombination we have chosen to focus on a key early step(s): homologous pairing and strand exchange between homologous parental DNAs. A fundamental problem in homologous recombination is how the search for homology between the two DNAs is carried out. In all current models a homologous recombination protein, such as the prototypical E. coli RecA protein, loads onto a single-strand DNA generated from one duplex DNA and scans another duplex to form a synaptic (pairing) complex. Eventually, DNA strands are exchanged and a new heteroduplex is formed. While homologous pairing and strand exchange are the earliest contacts between two parental DNAs mediated by RecA and its eukaryotic homologues, Rad51 and Dmc1, homologous recombination is initiated at DNA double-strand breaks (DSBs). The protein that catalyzes DSB formation in meiosis in the budding yeast, Saccharomyces cerevisae, is the product of the SPO11 gene. Surprisingly, Spo11 homologues are dispensable for synapsis in C. elegans and D. melanogaster yet required for meiotic recombination. We have generated a SPO11 mouse knock-out to investigate the biological function of this gene in mammals. Disruption of mouse SPO11 results in infertility. Spermatocytes arrest prior to pachytene with little or no homologous synapsis and undergo apoptosis. Recently, we have been conducting DNA microarray experiments to determine those meiotically expressed genes whose expression is modified by a DSB. In young mice, before degenerative changes have set in as a result of the apoptosis seen in the knockouts, there are only a few dozen genes that are differentially expressed in Spo11 -/- compared to wild type. Among the genes most affected are the Hop2 and Mnd1 genes. These are homologues of genes that affect homologous pairing in yeast. We have generated a knockout of the Hop2 gene in the mouse. Its meiotic phenotype shows a profound meiotic arrest that is unlike any seen previously. Unlike most knockouts with a meiotic phenotype these mice show no synapsis of any kind. That is, whereas most knockouts, show some willy-nilly non-homologous synapsis, spermatocytes from these mice are arrested without almost any synapsis. The chromosomes are somewhat compacted and appear normally decorated with both Rad51 and Dmc1, as if they are on the cusp of synapsis but fail to proceed forward. This finding suggests that the Hop2 protein might play a heretofore-unrecognized central role in bringing meiotic chromosomes together. Prompted by this biologically inspired hypothesis we have purified and studied the biochemical properties of the 25 Kda Hop2 protein. Most gratifyingly, we have been able to show that Hop2 protein can promote the strand invasion (D-loop formation) and strand exchange reactions characteristic of bona fide homologous recombination proteins. Remarkably, with respect to the very important initiating strand invasion activity it is more active than either RecA homologue; Dmc1, mediates strand invasion poorly and Rad51, mediates strand invasion only in the presence of the Rad54 protein. Finally, we have been able to show that Hop2 can act as an accessory protein for the strand exchange carried out by Rad51. Thus, Hop2 is not only a true novel recombinase but also may be the crucial and central protein in initiating interhomolog contacts in meiotic HR. In all organisms, homologous recombination is inextricably related to DNA repair and replication, hence cell proliferation and its control. Finally, we have used whole-genome cDNA arrays were used to analyze changes in the levels of gene expression of all E. coli ORFs after treatment with mitomycin C (MMC). Several experiments, which differ in the mode of MMC treatment, were performed, and expression profiles of E. coli cells at different time points after the addition of the DNA damaging agent were analyzed. As a whole, these experiments consist of 16 different hybridizations corresponding to about 70,000 individual data points. Around 5-10% of all genes show significant changes in their level of expression. As shown before, the expression level of several LexA-regulated genes was increased after DNA damage. On the other hand, most of those genes that show significant changes in their level of expression have not been shown previously to be inducible or repressed in the process of DNA repair. An attempt was made to classify all genes based on their responses to DNA damage. Using cluster analysis of the gene expression data it is possible to divide all the genes into at least 12 different clusters. Of the 400 or so upregulated genes about 100 were poorly annotated or not annotated at all. Of these 100 we have selected about 50 that encode for proteins of modest size, are not clearly membrane proteins and show some evolutionary conservation. We have made gene deletion strains for most of these genes and are now studying their phenotypes, both with regard to DNA metabolism (in collaboration with Sue Lovett at Brandeis) and general intermediate metabolism using the Biolog phenotypic arrays. In addition, we have initiated a structural genomics project (in collaboration with Galya Obmolova, Alex Teplyakov and Gary Gilliland at CARB) to determine the structure of as many as possible of the protein products of these 50 genes. So far we have obtained the molecular structure of six of these proteins. Understanding the evolutionary selective forces that fashioned the sex chromosomes from a putative ancestral autosome pair is a major problem in biology. In the last couple of years it has been reported that in C. elegans and D. melanogaster (Parisi et al. (2003) Science 299, 697) male-specific genes are underrepresented on the X-chromosome whereas the opposite is true in the mouse. Is the mouse really different? Our analysis of both EST and Affymetrix expression databases for different tissues indicates that testis-enriched and male-biased genes are significantly underrepresented on the mouse X-chromosome. Why the discrepancy between these results and the previous mouse study? Our analysis of the Spo11 adult mice microarray data provides a time line for the expression of testis genes. In effect, the Spo11 -/- adult testes represent a partial castration, that is, they are depleted for all cells after the arrest in meiosis I and enriched for earlier cells. We find that those genes expressed in late cells, including the majority of testis-enriched genes, are underrepresented on the X. However, early genes are overrepresented. These results reconcile all the data since the previously published mouse data pertained to early spermatogonial genes only.