Dna2 helicase/nuclease causes replicative fork stalling and double-strand breaks in the ribosomal DNA of Saccharomyces cerevisiae.
We have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae and contributes to the shortened lifespan of dna2 mutants. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We show directly that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the early aging, hypomorphic dna2-2 helicase mutant. Deletion of FOB1, encoding the fork barrier protein, suppresses the elevated pausing and DSB formation, and represses initiation at rDNA ARSs. The dna2-2 mutation is synthetically lethal with deltarrm3, encoding another DNA helicase involved in rDNA replication. It does not appear to be the case that the rDNA is the only determinant of genome stability during the yeast lifespan however since strains carrying deletion of all chromosomal rDNA but with all rDNA supplied on a plasmid, have decreased rather than increased lifespan. We conclude that the replication-associated defects that we can measure in the rDNA are symbolic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.
Weitao
Tao
T
Braun Laboratories 147-75, California Institute of Technology, Pasadena, California 91125, USA.
Budd
Martin
M
Hoopes
Laura L Mays
LL
Campbell
Judith L
JL
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001761:Bleomycin / Q000494:pharmacology; D004249:DNA Damage; D004265:DNA Helicases / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics*; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion
D004271:DNA, Fungal; D004275:DNA, Ribosomal; D029701:Saccharomyces cerevisiae Proteins; D001761:Bleomycin; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae