The DNA Damage Response Pathway Contributes to the Stability of Chromosome III Derivatives Lacking Efficient Replicators

Abstract

In eukaryotic chromosomes, DNA replication initiates at multiple origins. Large inter-origin gaps arise when several adjacent origins fail to fire. Little is known about how cells cope with this situation. We created a derivative of Saccharomyces cerevisiae chromosome III lacking all efficient origins, the 5ORIΔ-ΔR fragment, as a model for chromosomes with large inter-origin gaps. We used this construct in a modified synthetic genetic array screen to identify genes whose products facilitate replication of long inter-origin gaps. Genes identified are enriched in components of the DNA damage and replication stress signaling pathways. Mrc1p is activated by replication stress and mediates transduction of the replication stress signal to downstream proteins; however, the response-defective mrc1AQ allele did not affect 5ORIΔ-ΔR fragment maintenance, indicating that this pathway does not contribute to its stability. Deletions of genes encoding the DNA-damage-specific mediator, Rad9p, and several components shared between the two signaling pathways preferentially destabilized the 5ORIΔ-ΔR fragment, implicating the DNA damage response pathway in its maintenance. We found unexpected differences between contributions of components of the DNA damage response pathway to maintenance of ORIΔ chromosome derivatives and their contributions to DNA repair. Of the effector kinases encoded by RAD53 and CHK1, Chk1p appears to be more important in wild-type cells for reducing chromosomal instability caused by origin depletion, while Rad53p becomes important in the absence of Chk1p. In contrast, RAD53 plays a more important role than CHK1 in cell survival and replication fork stability following treatment with DNA damaging agents and hydroxyurea. Maintenance of ORIΔ chromosomes does not depend on homologous recombination. These observations suggest that a DNA-damage-independent mechanism enhances ORIΔ chromosome stability. Thus, components of the DNA damage response pathway contribute to genome stability, not simply by detecting and responding to DNA template damage, but also by facilitating replication of large inter-origin gaps.

Rtt107 Is a Multi-functional Scaffold Supporting Replication Progression with Partner SUMO and Ubiquitin Ligases

“…3) lacking a portion of the right arm (referred to as Chr3ΔR, 170 kb) can be maintained in cells containing a normal Chr. 3 (Theis et al, 2007, 2010) (Figure 7A). One variant has 12 ARSs and the other has 7 ARSs, and are referred to as Chr3ΔR-12ARS and Chr3ΔR-7ARS, respectively.…”

“…One variant has 12 ARSs and the other has 7 ARSs, and are referred to as Chr3ΔR-12ARS and Chr3ΔR-7ARS, respectively. The extra five ARSs in the former are known to fire, and thus greatly reduce replicon sizes (Theis et al, 2007, 2010). Using PFGE coupled with Southern blot analysis, fully replicated Chr.…”

Behavior of replication origins in Eukaryota – spatio-temporal dynamics of licensing and firing

“…Supporting experimental evidence also shows that for a chromosome with a 160 kb origin-less telomeric region, the rate of loss is more than 20 times higher than in the same region within a chromosome body. 35 Yet, this effect appears to be smaller than theoretically predicted, which may imply that some additional mechanism exists to enhance telomeric replication. It still consistently confirms the thesis of origin distribution being determined by the risk of stall events as one of major factors.…”

“…The chromosome with an origin-less region of approximately 160 kb (close to the calculated maximum gap) had a 3 times higher loss rate compared to the correct value. 35 This overall protection against fork stalling does not reflect dormancy or activity of origins, but rather the total number of licensed ROs.…”

The contribution of dormant origins to genome stability: From cell biology to human genetics

“…Large inter-origin distances should therefore be avoided, and in all 5 yeasts examined ( S. cerevisiae , S. pombe , Kluyveromyces lactis , Lachancea kluyveri and Lachancea waltii ) this is clearly the case. Indeed, previous work [75] had shown that deletion of five origins in S. cerevisiae , creating a large inter-origin distance of 160 kb (close to the expected value of the largest inter-origin distance if origins were randomly distributed), resulted in an increased chromosome loss rate exactly in line with the increased probability of double fork stalls [57]. In order to globally minimize large gaps and the probability of double fork stalls, it is also optimal to position replication origins at regularly spaced intervals across the genome.…”