Genetic recombination is a critical process in the germline and soma of higher eukaryotes. During meiosis, homologous recombination (HR) is needed for proper chromosome segregation and genetic diversification. In somatic ceils, HR is critical for repairing certain types of DNA damage, namely double strand breaks (DSBs). Failure to repair such damage accurately can lead to deleterious chromosomal rearrangements, mutation, or cell death. This proposal centers on the genetic control of recombination and maintenance of genome integrity in the mouse. In the previous grant period, novel mutagenesis screens were conducted to create mutations disrupting meiosis (in germ cells) or genomic stability (in somatic cells). Experiments are proposed to conduct functional and genetic studies on two mutations affecting key steps in meiotic HR (Mei1 and mei4), and two causing genomic instability (GIN; Chaos1 and Chaos3). Mei1 is a novel, vertebrate-specific gene affecting the key step of recombination initiation, induction of DSBs. Its role in DSB formation will be investigated by cytological studies of meiotic chromosomes, functional characterization of interacting proteins, and direct assessment of DSB formation at recombination hotspots. mei4 mutants lack chiasmata, thereby disrupting chromosome segregation at MI. It maps to a region not containing known recombination genes, so positional cloning will identify a novel molecule needed for crossing over. The two GIN mutations were identified using a micronucleus assay, followed by positional cloning. Chaos1 is radiation- and crosslink-sensitive allele of Polq, an ortholog of Drosophila mus308 (mutagen sensitive 308) encoding both helicase and polymerase domains. POLQ may be involved in recombinational repair of damaged replication forks. Neonatal synthetic lethality and severe runting is induced in most Chaos1/Atm double mutants, but lymphoma latency is increased in survivors. The basis for these effects on tumor progression and cell proliferation will be explored. Chaos3 is likely a semidominant allele of the replication licensing complex protein Mcm4. Early data indicate these mice are tumor prone, potentially representing the first direct relationship between a mutant replication licensing gene and cancer. Mutant mice and cells will be characterized with respect to proliferation, cancer susceptibility and tumor spectrum. ? ?
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