Recombination and DNA Break Repair: Mechanism and Control

Genetic recombination plays a crucial role in the maintenance of chromosomal integrity and the generation of genetic diversity. During mitotic growth of cells, faithful repair of DNA double-strand breaks (DSBs) requires homologous recombination. Failure to repair DSBs is often lethal, as essential genes can be lost. During meiosis, recombination plays an important role in the proper segregation of chromosomes and the formation of viable sex cells. Aberrancies in recombination thus produce chromosomal losses and rearrangements, such as deletions and translocations, and can result in birth defects or cancers. Understanding the molecular mechanism of recombination will give us insight into the causes of these diseases and possibly ways of predicting or preventing them; it will also help create new cell lines and mutant organisms by gene targeting. Common features of recombination in model organisms, including easily studied microorganisms, aid identifying human genes for recombination and DSB repair, which may be altered in specific diseases such as cancer.

Our lab's goals are to elucidate how recombination and DSB repair are accomplished and how they are regulated to occur at the proper place and time. Our research is focused on meiotic recombination in the fission yeast Schizosaccharomyces pombe and on the major (RecBCD) pathway of recombination in the bacterium Escherichia coli. In both organisms we approach this problem genetically, by analyzing mutants altered in the process, and biochemically, by studying the enzymes and special DNA sites (hotspots) that promote recombination and repair. These approaches are greatly facilitated by the advanced genetics and biochemistry of these microorganisms.

We are studying the complex RecBCD enzyme and its control by the recombination hotspot Chi (5' GCTGGTGG 3'). RecBCD has multiple activities on DNA, including DNA unwinding and DNA hydrolysis. Using mutant RecBCD enzymes and electron microscopy, we have found that RecBCD unwinds DNA by producing a growing ss DNA loop through the combined action of a fast helicase (RecD) and a slower ss DNA translocase (RecB). Upon encountering Chi, RecBCD enzyme is changed such that it produces a 3' ss DNA end onto which it loads RecA strand-exchange protein; the physical basis of Chi's regulation of RecBCD is unknown. We have recently isolated novel classes of recBCD mutants whose properties lead us to propose a "signal transduction cascade" model, in which RecC recognizes Chi and signals RecD to stop unwinding DNA; RecD then signals RecB to cut the DNA and to begin loading RecA. We are testing the predictions of this model with a combination of genetics and enzymology. RecBCD is an excellent example of a complex "protein machine"; understanding the mechanism and control of RecBCD will aid studies of other such machines, including those acting in replication and transcription.

Meiotic Recombination in S. pombe

We have recently outlined a pathway of proteins promoting meiotic recombination, which we divide into three stages: chromosome movement and pairing (called "horsetailing" and "bouquet" formation), DSB formation, and DSB repair. We have placed dozens of gene products into these stages and identified two central DNA intermediates of recombination - DSBs and Holliday junctions (HJs). Our current genome-wide analysis of DSBs confirms our previous mapping of DSBs to hotspots of recombination; these results will help us find the chromosomal determinants of these hotspots. In addition, there is a paucity of DSBs in intervals that nevertheless have abundant crossing-over, a result suggesting that lesions other than DSBs can initiate recombination. Single HJs predominate instead of the double HJs seen in budding yeast. Our current research has revealed additional ways in which recombination differs in these two yeasts. For example, we have found roles for RNAi components, not present in budding yeast, and a histone modifying enzyme in the repression of DSB formation at recombinationally silent centromeres. We have found that palindromic DNA, a frequent feature in the human genome, confers a recombination hotspot independent of the major DSB-forming protein Rec12 (Spo11 homolog); we are determining if palindromes promote non-homologous translocations, as they do during human meiosis. Determining the mechanism of homologous and non-homologous recombination in S. pombe will aid studies of the mechanism in human cells.