investigator's application): Cells respond to DNA damage by both arresting the cell cycle and inducing the transcription of genes whose products facilitate DNA repair. The biochemical pathways that ensure this co-ordination are called checkpoints. The long range goal of the studies is to understand how eukaryotic cells sense and respond to DNA replication blocks and DNA damage, using the RNR (ribonucleotide reductase) genes of S. cerevisiae as models. The roles of the SAD, POL, DUN, and CRT checkpoint genes in the DNA damage-induced response will be studied by genetic, molecular, and biochemical means. DNA polymerase epsilon, which is encoded by POL2, is a candidate sensor of DNA damage and replication blocks that controls the S phase checkpoint. The relationship between the polymerase and checkpoint functions of POL2 will be studied to determine how the polymerase senses replication problems. Proteins that co-operate with POL2 to transmit its cell cycle arrest signals will be identified by genetic and physical methods. SAD1 encodes an essential protein kinase that is a central transducer of the DNA damage signal, controlling the G1, S, and G2 checkpoints and transcriptional response through Dun1 kinase. The regulation of Sad1 by DNA damage will be determined, and Sad1 substrates will be identified. Attempts will be made to determine the essential function of Sad1, and additional genes in the Sad1 pathway will be identified through genetic means. The goal is to determine how the cell cycle is arrested and whether arrest proceeds through Cdk inhibitor proteins. The SAD4 and SAD6 genes will be studied to determine how they link SAD1 function to the DNA damage pathway. Transcriptional activation of DNA damage inducible genes requires activation of the Dun1 kinase. Downstream of Dun1 is a gene (CRT1) whose product represses damage inducible genes. The Sad1 kinase is upstream of Dun1. Biochemical and genetic analysis will be performed with Dun1 to determine whether it is directly activated by Sad1 and whether it functions to inactivate the CRT1 repressor. Genetic epistasis analysis will be performed between crt and dun mutants to order the DUN and CRT genes in the transcriptional induction pathway. Potential Dun1 substrates will be identified by genetic and immunological methods.
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