Chromosomal double strand breaks (DSBs) are cytotoxic lesions that occur spontaneously during normal cell metabolism or by treatment of cells with DNA-damaging agents. If unrepaired or repaired inappropriately, DSBs can lead to mutagenic events, such as chromosome loss, deletions, duplications or translocations, events that can lead to carcinogenesis. The repair of DSBs by homologous recombination (HR) relies on the presence of a homologous duplex to template repair of the broken chromosome and is generally considered to be an error-free mechanism. However, HR can lead to a local loss of heterozygosity (LOH) if the recombining sequences are not identical, and to extensive LOH if repair is associated with a crossover between chromosome homologs. Furthermore, if a repeated sequence at an ectopic site is utilized as the sequence donor and recombination is associated with crossing over, translocations can occur. When both ends of the DSB share homology with the donor duplex sequence, HR proceeds by a two-ended mechanism resulting in primarily non-crossover products. However, if coordination of the two ends is not maintained or only one end of the break is available, such as at a critically short telomere, repair can occur by break-induced replication (BIR). In this case, following strand invasion replication can extend for more than 100 kb to reach the end of the chromosome. This can cause extensive LOH or non-reciprocal translocation if invasion occurs at a dispersed repeated sequence. In this proposal we will continue mechanistic studies to understand how LOH and chromosome rearrangements occur by BIR or by resolution of recombination intermediates using the yeast model system.
The specific aims are: (1) A new system to monitor BIR repair of a chromosomal DSB will be used to determine the mechanism of DNA synthesis and test the idea that destabilization of the ssDNA intermediate decreases BIR efficiency. In addition, we plan to use the iPOND method to identify new factors involved in BIR by association with nascent DNA strands. (2) We will establish a new assay to detect template switching between artificial or natural repeats and identify genes that regulate this process. (3) We will determine the consequences of mis-regulation of structure-selective nucleases on DSB induced and spontaneous mitotic crossovers, and follow the fate of unresolved recombination intermediates during mitosis. The roles of mismatch repair, Rad1-Rad10 and the Mph1 helicase in controlling crossovers will also be determined.
The repair of DNA double-strand breaks by break-induced replication (BIR) can lead to several types of chromosome rearrangements that are associated with human disease. BIR is also involved in maintaining telomere length in the absence of telomerase and this process is activated in some human tumors. In this proposal, genetic and physical approaches will be used to define the mechanism used for DNA synthesis during BIR and to identify the genes that regulate chromosome rearrangements.
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