Mrc1, Tof1 and Csm3 inhibit CAG.CTG repeat instability by at least two mechanisms
David F Razidlo1, Robert S Lahue
1Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Box 986805, Omaha, NE 68198-6805, USA.
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.
Insights
Researchers identified Mrc1 (human Claspin homolog) and its partners Tof1 and Csm3 as key inhibitors of trinucleotide repeat instability. These proteins utilize distinct mechanisms to prevent both contraction and expansion of repetitive DNA sequences.
Area of Science:
- Molecular Biology
- Genetics
- DNA Repair
Background:
- Trinucleotide repeats are prone to expansion and contraction in various organisms.
- Previous studies identified protein factors modulating repeat instability through candidate gene or mutant screens.
- A need existed to identify factors that specifically inhibit repeat contraction.
Purpose of the Study:
- To identify Saccharomyces cerevisiae mutants exhibiting increased CAG.CTG repeat contraction rates.
- To elucidate the mechanisms by which Mrc1, Tof1, and Csm3 regulate trinucleotide repeat instability.
- To differentiate the roles of replication and DNA damage checkpoints in repeat dynamics.
Main Methods:
- Utilized a Saccharomyces cerevisiae disruption library to screen for mutants with higher CAG.CTG repeat contraction rates.
- Performed genetic analysis to assess the impact of Mrc1, Tof1, Csm3, and checkpoint protein deficiencies on repeat instability.
- Investigated the role of protein coupling activity in preventing single-strand DNA accumulation and secondary structure formation.
Main Results:
- Mrc1, Tof1, and Csm3 were identified as novel inhibitors of CAG.CTG repeat contraction.
- Loss of Mrc1, Tof1, or Csm3 significantly elevated contraction rates of (CAG)(20) tracts.
- Deficiencies in Mrc1, Tof1, Csm3, and several DNA damage checkpoint proteins elevated expansion rates of (CTG)(13) tracts, suggesting checkpoint involvement in inhibiting expansions.
Conclusions:
- Mrc1, Tof1, and Csm3 inhibit trinucleotide repeat instability through at least two distinct mechanisms.
- Protein coupling activity of Mrc1, Tof1, and Csm3 appears crucial for preventing repeat contractions.
- The DNA damage checkpoint plays a role in inhibiting repeat expansions after the formation of repeat-dependent structures.
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