This proposal focuses on the steps of DNA replication that are important in maintaining the stability of long tracts of CAG trinucleotide repeats. Long CAG repeat tracts are found in some human genes and are the mutational cause of over a dozen inherited neurological disorders. Using yeast mutants that are defective in DNA replication, we have defined the replication steps that are most likely to lead to tract expansions and to those that give rise to tract contractions. The studies described in this proposal further elaborate the mechanisms of tract expansion and tract contraction. An initial study examines whether Okazaki fragments containing CAG repeats are the same length as fragments containing unique sequence DNA. By detailed studies on mutations of the DNA helicase, DNA polymerase, DNA ligase, flap endonuclease and proliferating cell nuclear antigen (PCNA) we will define the mechanism by which flaps of DNA give rise to tract expansions. Additional studies look at whether tract contractions are limited by the extent to which the replication fork opens and by the presence of topoisomerases. Finally, we will examine whether chromosomes bearing CAG repeat tracts are lost as a result of replication stalling.
