Mrc1, Tof1 and Csm3 inhibit CAG·CTG repeat instability by at least two mechanisms

Abstract

Trinucleotide repeats frequently expand and contract in humans and model organisms. Protein factors that modulate this process have been found by candidate gene approaches or mutant screens for increased expansion rates. To extend this effort, Saccharomyces cerevisiae mutants with higher CAG CTG repeat contraction rates were sought using a disruption library. This screen identified Mrc1, the homolog of human Claspin, which mediates the replication and DNA damage checkpoints, and also couples the replicative helicase and polymerase. Genetic analysis showed that Mrc1, along with Tof1 and Csm3, inhibits instability in two distinct ways. Contraction rates of (CAG) 20 tracts are elevated by loss of Mrc1, Tof1 or Csm3, but not by defects in most replication checkpoint or DNA damage checkpoint proteins. The three proteins likely inhibit contractions primarily through their coupling activity, which would prevent accumulation of single-strand template DNA prior to the formation of aberrant secondary structure. In contrast, expansion rates of (CTG) 13 are elevated in strains defective for Mrc1, Tof1, Csm3, Mec1, Ddc2, Rad24, Ddc1, Mec3, Rad17, Rad9, Rad53 or Chk1, suggesting that the DNA damage checkpoint inhibits expansions after formation of repeat-dependent structures. Together, these results indicate that at least two Mrc1-dependent mechanisms function to reduce CAG•CTG repeat instability.

Expansions, contractions, and fragility of the spinocerebellar ataxia type 10 pentanucleotide repeat in yeast

“…This effect of the Tof1 knockout on the expansions of a pentanucleotide repeat is quantitatively similar to its effect on trinucleotide repeats (17,29). Thus, functional Tof1 protein precludes expansions of various unstable repeats.…”

The Intra-S Phase Checkpoint Protein Tof1 Collaborates with the Helicase Rrm3 and the F-box Protein Dia2 to Maintain Genome Stability in Saccharomyces cerevisiae

“…It has been reported that TOF1 and CSM3 can cause contraction of (CNG) n trinucleotide repeats and TOF1 along with several checkpoint-related proteins can cause trinucleotide repeat expansion (44). It is possible that such a mechanism could account for the observed changes in telomere length.…”

Altered Replication in Human Cells Promotes DMPK (CTG)n · (CAG)n Repeat Instability

“…Previous results demonstrated that DNA hairpins form in a replicationdependent manner at the ectopic TNRs and that DNA replication origin activity increased the instability of the adjacent hairpinprone TNRs in vivo (56). Since expanded (CTG) n · (CAG) n TNRs are sites of replication fork stalling and genome instability in yeast (30,73,89,90), we tested the effect of (CTG) n · (CAG) n TNRs on replication fork progress and the possible requirement for stabilization of TNR replication by the fork protection proteins Timeless, Tipin, and Claspin. We found that replication forks were stalled by flanking expanded TNRs and that TNR stability was dramatically decreased after knockdown of the replication-forkstabilizing proteins.…”