Checkpoint inhibition of origin firing prevents DNA topological stress

Abstract

A universal feature of DNA damage and replication stress in eukaryotes is the activation of a checkpoint-kinase response. In S-phase, the checkpoint inhibits replication initiation, yet the function of this global block to origin firing remains unknown. To establish the physiological roles of this arm of the checkpoint, we analyzed separation of function mutants in the budding yeast Saccharomyces cerevisiae that allow global origin firing upon replication stress, despite an otherwise normal checkpoint response. Using genetic screens, we show that lack of the checkpoint-block to origin firing results in a dependence on pathways required for the resolution of topological problems. Failure to inhibit replication initiation indeed causes increased DNA catenation, resulting in DNA damage and chromosome loss. We further show that such topological stress is not only a consequence of a failed checkpoint response but also occurs in an unperturbed S-phase when too many origins fire simultaneously. Together we reveal that the role of limiting the number of replication initiation events is to prevent DNA topological problems, which may be relevant for the treatment of cancer with both topoisomerase and checkpoint inhibitors.

Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phase

“…If the checkpoint-mediated inhibition of G1/S transcription and Sld3/Dbf4 both contribute to prevent precocious S-phase entry then we hypothesised that loss of both pathways should show synthetic lethality in the presence of DNA damage. We have previously conducted genetic interaction analysis of the sld3-A/dbf4-A alleles with the yeast whole genome gene knock-out collection in the presence of the DNA damaging agent phleomycin (Morafraile et al, 2019). Significantly loss of function of genes that result in a delay in the G1/S transition, such as CLN2, SWI4 and BCK2 (Di Como et al, 1995) Here we show that the two critical targets of the S-phase checkpoint mediated inhibition of origin firing, Sld3 and Dbf4, are actually regulated by Rad53 after DNA damage throughout the cell cycle (Figures 1 and 2).…”

“…Therefore, multiple mechanisms exist to ensure the robust inhibition of replication initiation in S-phase by direct targeting of Sld3/Treslin and Dbf4-dependent kinase and by inhibition of CDK activity (Lanz et al, 2019). One function of such robust inhibition of origin firing during S-phase in the presence of DNA lesions is to prevent the exhaustion of essential factors, such as topoisomerase activities, by excessive numbers of replisomes (Morafraile et al, 2019;Toledo et al, 2017).…”

Supercoiling, R-Loops, Replication and the Functions of Bacterial Type 1A Topoisomerases

“…Interestingly, it has been recently shown in yeast that the role of limiting replication initiation via the checkpoint-kinase response induced by DNA damage, is to prevent topological stress that leads to increased DNA catenation, followed by DNA damage and chromosome loss [97]. Topological stress is also observed in non-stressed S-phase cells when too many replication origins are simultaneously activated.…”

Checkpoint inhibition of origin firing prevents inappropriate replication outside of S-phase

“…If the checkpoint-mediated inhibition of G1/S transcription and Sld3/Dbf4 both contribute to prevent precocious S-phase entry then we hypothesised that loss of both pathways should show synthetic lethality in the presence of DNA damage. We have previously conducted genetic interaction analysis of the sld3-A/dbf4-A alleles with the yeast whole genome gene knock-out collection in the presence of the DNA damaging agent phleomycin (Morafraile et al, 2019). Significantly loss of function of genes that result in a delay in the G1/S transition, such as CLN2, SWI4 and BCK2 (Di Como et al, 1995) improved the growth of sld3-A/dbf4-A in the presence of phleomycin (suppressors, Supplementary Figure 5), whereas loss of function of genes that would result in the acceleration of G1/S, such as WHI5 and SIC1 (Bertoli et al, 2013) were synthetic sick with the sld3-A/dbf4-A alleles (enhancers, Supplementary Figure 5).…”

“…The checkpoint-mediated inhibition of origin firing likely occurs by similar mechanisms in human cells as the checkpoint kinases also bind to and inhibit the Sld3 orthologue Treslin (Boos et al, 2011;Guo et al, 2015) and inhibit DDK (Costanzo et al, 2003;Lee et al, 2012). One function of inhibiting origin firing during S-phase in the presence of DNA lesions is to prevent the exhaustion of essential factors, such as topoisomerase activities, by excessive numbers of replisomes (Morafraile et al, 2019;Toledo et al, 2017).…”