The yeast checkpoint factors Mrc1p and Tof1p travel with the replication fork and mediate the activation of the Rad53p kinase in response to a replication stress. We show here that both proteins are required for normal fork progression but play different roles at stalled forks. Tof1p is critical for the activity of the rDNA replication fork barrier (RFB) but plays a minor role in the replication checkpoint. In contrast, Mrc1p is not necessary for RFB activity but is essential to mediate the replication stress response. Interestingly, stalled forks did not collapse in mrc1Delta cells exposed to hydroxyurea (HU) as they do in rad53 mutants. However, forks failed to restart when mrc1Delta cells were released from the block. The critical role of Mrc1p in HU is therefore to promote fork recovery in a Rad53p-independent manner, presumably through the formation of a stable fork-pausing complex.
The members of the RecQ family of DNA helicases play conserved roles in the preservation of genome integrity. RecQ helicases are implicated in Bloom and Werner syndromes, which are associated with genomic instability and predisposition to cancers. The human BLM and WRN helicases are required for normal S phase progression. In contrast, Saccharomyces cerevisiae cells deleted for SGS1 grow with wild-type kinetics. To investigate the role of Sgs1p in DNA replication, we have monitored S phase progression in sgs1D cells. Unexpectedly, we ®nd that these cells progress faster through S phase than their wild-type counterparts. Using bromodeoxyuridine incorporation and DNA combing, we show that replication forks are moving more rapidly in the absence of the Sgs1 helicase. However, completion of DNA replication is strongly retarded at the rDNA array of sgs1D cells, presumably because of their inability to prevent recombination at stalled forks, which are very abundant at this locus. These data suggest that Sgs1p is not required for processive DNA synthesis but prevents genomic instability by coordinating replication and recombination events during S phase.
Accurate and complete DNA replication is fundamental to maintain genome integrity. While the mechanisms and underlying machinery required to duplicate bulk genomic DNA are beginning to emerge, little is known about how cells replicate through damaged areas and special chromosomal regions such as telomeres, centromeres, and highly transcribed loci . Here, we have investigated the role of the yeast cullin Rtt101p in this process. We show that rtt101Delta cells accumulate spontaneous DNA damage and exhibit a G(2)/M delay, even though they are fully proficient to detect and repair chromosome breaks. Viability of rtt101Delta mutants depends on Rrm3p, a DNA helicase involved in displacing proteinaceous complexes at programmed pause sites . Moreover, rtt101Delta cells show hyperrecombination at forks arrested at replication fork barriers (RFBs) of ribosomal DNA. Finally, rtt101Delta mutants are sensitive to fork arrest induced by DNA alkylation, but not by nucleotide depletion. We therefore propose that the cullin Rtt101p promotes fork progression through obstacles such as DNA lesions or tightly bound protein-DNA complexes via a new mechanism involving ubiquitin-conjugation.
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