Tuesday, November 26, 2024
11/26/2024
DESCRIPTION (provided by applicant): The Ty1 retroelement of yeast transposes via a mechanism bearing many similarities to retroviruses which infect larger eukaryotes. The Ty1 transposition process is inherently temperature sensitive. In a screen of the Saccharomyces deletion collection we identified RFX1 and SML1 as inhibitors of high temperature Ty1 transposition. RFX1 and SML1 are both negative regulators of ribonucleotide reductase (RNR), the rate limiting enzyme in the production of dNTPs. An adequate supply of dNTPs is essential for cellular division and DNA repair, and RNR is regulated by both of these processes. DNA repair is tightly regulated, critical for maintaining genome stability, and defects in DNA repair, as well as elevated RNR activity, is seen in some types of human cancers. The transposition process generates double stranded DNA molecules that are then integrated or recombined into the genome, a process that has been shown to involve components of the DNA repair pathway. We have identified two additional genes that regulate Ty1 mobility by inhibiting recombination of Ty1 cDNA. RFX1 and SML1 are effectors of the central Mec1 DNA repair pathway. We propose to use genetic analysis to determine the interactions of RFX1 and SML1 with other members of the Mec1 pathway in regulating Ty1 mobility. The Rad53 transducer, which acts downstream of Mec1, is an important mediator of this pathway. We will use phosphorylation of the Rad53 protein as a marker for induction of the Mec1 repair pathway during transposition. We will also attempt to determine whether Ty1 DNA sequences are recognized differently than other sequences by the cellular homologous recombination pathway. Finally, Southern analysis will be used to determine the stability and recombination targets of Ty1 cDNA molecules in various mutant strains, and the role of Ty1 integrase in these processes.
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