Mus81 functions in the quality control of replication forks at the rDNA and is involved in the maintenance of rDNA repeat number in Saccharomyces cerevisiae.
Previous studies in yeast have suggested that the SGS1 DNA helicase or the Mus81-Mms4 structure-specific endonuclease is required to suppress the accumulation of lethal recombination intermediates during DNA replication. However, the structure of these intermediates and their mechanism of the suppression are unknown. To examine this reaction, we have isolated and characterized a temperature-sensitive (ts) allele of MUS81. At the non-permissive temperature, sgs1Deltamus81(ts) cells arrest at G(2)/M phase after going through S-phase. Bulk DNA replication appears complete but is defective since the Rad53 checkpoint kinase is strongly phosphorylated under these conditions. In addition, the induction of Rad53 hyper-phosphorylation by MMS was deficient at permissive temperature. Analysis of rDNA replication intermediates at the non-permissive temperature revealed elevated pausing of replication forks at the RFB in the sgs1Deltamus81(ts) mutant and a novel linear structure that was dependent on RAD52. Pulsed-field gel electrophoresis of the mus81Delta mutant revealed an expansion of the rDNA locus depending on RAD52, in addition to fragmentation of Chr XII in the sgs1Deltamus81(ts) mutant at permissive temperature. This is the first evidence that Mus81 functions in quality control of replication forks and that it is involved in the maintenance of rDNA repeats in vivo.
Ii
Miki
M
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, United States. mikiii@mac.com
Ii
Tatsuya
T
Brill
Steven J
SJ
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D064447:Checkpoint Kinase 2; D004260:DNA Repair; D004261:DNA Replication / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D016521:Electrophoresis, Gel, Pulsed-Field; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism*; D005800:Genes, Fungal; D008957:Models, Genetic; D009154:Mutation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics / Q000378:metabolism; D053484:RecQ Helicases / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D004720:Endonucleases; C411624:MUS81 protein, S cerevisiae; C090472:SGS1 protein, S cerevisiae; D053484:RecQ Helicases