S-phase checkpoint regulations that preserve replication and chromosome integrity upon dNTP depletion

Abstract

DNA replication stress, an important source of genomic instability, arises upon different types of DNA replication perturbations, including those that stall replication fork progression. Inhibitors of the cellular pool of deoxynucleotide triphosphates (dNTPs) slow down DNA synthesis throughout the genome. Following depletion of dNTPs, the highly conserved replication checkpoint kinase pathway, also known as the S-phase checkpoint, preserves the functionality and structure of stalled DNA replication forks and prevents chromosome fragmentation. The underlying mechanisms involve pathways extrinsic to replication forks, such as those involving regulation of the ribonucleotide reductase activity, the temporal program of origin firing, and cell cycle transitions. In addition, the S-phase checkpoint modulates the function of replisome components to promote replication integrity. This review summarizes the various functions of the replication checkpoint in promoting replication fork stability and genome integrity in the face of replication stress caused by dNTP depletion.

Keywords: ATR/Mec1/Rad3; Chromosome fragility; Fork remodeling; Helicases; Nucleases; Rad53/CHK1/Cds1; Stalled replication forks.

Fig. 1

S-phase checkpoint-dependent replication fork-extrinsic controls in response to DNA replication inhibition. a Cellular controls that inhibit mitosis in the presence of stalled forks and incomplete DNA replication. b S-phase-dependent checkpoint signaling required for the up-regulation of dNTPs following DNA replication inhibition and DNA damage. c Molecular mechanisms underlying replication origin firing inhibition upon replication stress or blocked DNA synthesis (related to "Replication fork-extrinsic S-phase checkpoint-dependent regulations triggered by DNA replication inhibition"). d Checkpoint-mediated restriction of gene gating in budding yeast

Fig. 2

DNA replication fork alterations in checkpoint mutants and mechanisms contributing to proficient DNA replication. a DNA replication fork alterations (resected forks and reversed forks) accumulating in rad53 mutants of S. cerevisiae treated with hydroxyurea. The relative percentage of each major DNA replication fork intermediate is shown based on previously published results [64, 67]. b Replication stress induces uncoupling events between leading and lagging strands. Subsequent re-annealing of the parental and nascent strands can promote structural transitions at the stalled replication forks. Processing of the intermediates can also cause chromosome breakage. c Cellular mechanisms for fork stabilization and re-start. Re-priming coupled to DNA damage tolerance can preserve the normal DNA replication fork architecture. DNA replication inhibition and DNA lesions can induce fork uncoupling, formation of long ssDNA stretches, long DNA flaps and fork reversal. Activities that are potentially implicated in processing of flaps and reversed forks are shown (related to "Phenotypes caused by S-phase checkpoint dysfunction in unperturbed conditions and after dNTP depletion", "S-phase checkpoint roles in fork architecture: prevention of pathological DNA transitions or resolution of transient DNA intermediates?" and "S-phase checkpoint-dependent phosphorylation events at stalled replication forks" of the review)

Fig. 3

DNA substrates and protein factors required for S-phase checkpoint activation. a High amounts of ssDNA–RPA complexes at stalled forks and primer–template substrates can be induced by uncoupling of leading and lagging strand DNA synthesis, uncoupling between DNA polymerases and MCM DNA helicase, by discontinuous synthesis of the nascent strands, by hyper-priming activity of Polα, unwinding or resection of one of the nascent strands. b Simplified representation of the replication fork and some replisome components. Protein factors shown in yellow are involved in the activation of the Mec1^Rad3/ATR–Rad53 ^Cds1/CHK1 checkpoint pathway following dNTP deprivation. Physical and functional interactions instrumental to checkpoint activation are indicated through arrows and dashed lines, respectively (related to "Structural determinants and protein factors required for S-phase checkpoint activation in response to DNA replication stress")