Mutations in yeast replication proteins that increase CAG/CTG expansions also increase repeat fragility.
Expansion of trinucleotide repeats (TNRs) is the causative mutation in several human genetic diseases. Expanded TNR tracts are both unstable (changing in length) and fragile (displaying an increased propensity to break). We have investigated the relationship between fidelity of lagging-strand replication and both stability and fragility of TNRs. We devised a new yeast artificial chromomosme (YAC)-based assay for chromosome breakage to analyze fragility of CAG/CTG tracts in mutants deficient for proteins involved in lagging-strand replication: Fen1/Rad27, an endo/exonuclease involved in Okazaki fragment maturation, the nuclease/helicase Dna2, RNase HI, DNA ligase, polymerase delta, and primase. We found that deletion of RAD27 caused a large increase in breakage of short and long CAG/CTG tracts, and defects in DNA ligase and primase increased breakage of long tracts. We also found a correlation between mutations that increase CAG/CTG tract breakage and those that increase repeat expansion. These results suggest that processes that generate strand breaks, such as faulty Okazaki fragment processing or DNA repair, are an important source of TNR expansions.
Callahan
Julie L
JL
Department of Biology, Program in Genetics, Tufts University, Medford, Massachusetts 02155, USA.
Andrews
Kenneth J
KJ
Zakian
Virginia A
VA
Freudenreich
Catherine H
CH
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D001681:Biological Assay; D022201:Chromosomes, Artificial; D004249:DNA Damage; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism; D019915:DNA Primase / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D045585:Flap Endonucleases / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009154:Mutation; D015203:Reproducibility of Results; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D019680:Trinucleotide Repeat Expansion; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism
D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D019915:DNA Primase; D004258:DNA Polymerase III; D045585:Flap Endonucleases; D016914:Ribonuclease H; C078856:ribonuclease HI; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae; D011088:DNA Ligases