DNA repair mechanisms play an important role in maintaining genomic stability. Even though eukaryotic cells possess a large variety of means via which different types of lesions can be removed and repaired, a number of lesions persist in DNA into S phase; as a consequence, the progression of the replication fork is halted when the replisome encounters a lesion in the template strand. In the yeast Saccharomyces cerevisiae
, replication through DNA lesions is mediated by the Rad6-Rad18-dependent pathway, in which lesion bypass can occur by the action of translesion synthesis (TLS) DNA polymerases (Pols) (1
) or by a Rad5-Mms2-Ubc13 pathway that promotes lesion bypass by template switching (6
). A Rad6-Rad18-independent but Rad51-, Rad52-, and Rad54-dependent pathway can also promote fork progression through DNA lesions via template switching (9, 10).
In addition to DNA repair mechanisms that promote replication through DNA lesions, eukaryotic cells possess surveillance mechanisms called checkpoints that become activated when DNA replication is halted by DNA damage or by other perturbations that affect the progression of the replication fork. Intra-S-phase checkpoint, also known as replication checkpoint (11
), plays a crucial role in the maintenance of functional replication forks and in promoting cell survival and proliferation when cells are exposed to DNA-damaging agents (12
). In S. cerevisiae
, the key components of replication checkpoints are the Mec1 kinase and its downstream effector kinase Rad53. The recruitment of Mec1 to the stalled replication fork initiates the checkpoint pathway, where it phosphorylates and activates Rad53 (11
). Upon activation, this pathway affects many aspects of DNA replication that include the slowing of S-phase and cell cycle progression, downregulation of late origin firing, activation of DNA repair proteins, and stabilization of replication forks (11
The role of Mec1/Rad53 in the stabilization of replication forks is particularly important for maintaining the association of the replisome with the fork stalled at a DNA lesion. In the absence of checkpoint, the replisome dissociates and the stalled forks degenerate (12
). Thus, in mec1
Δ or rad53
Δ yeast cells treated with the alkylating agent methyl methanesulfonate (MMS), replication forks collapse irreversibly, leading to incomplete replication and cell death. This lethality occurs only when cells go through S phase in the presence of MMS and is not prevented by blocking the subsequent mitotic entry (12
). Such observations and a number of others (11
) have provided strong evidence that replication fork stabilization by Mec1 and Rad53 is critical for maintaining cell viability in yeast cells with damaged DNA and that checkpoint-dependent induction of transcription or regulation of late origin firing plays a relatively minor role (13
). However, how these checkpoint kinases prevent replisome dissociation is not known.
Since fork stabilization by the Mec1/Rad53-initiated pathway is essential for the maintenance of replication fork in yeast cells with damaged DNA, replication checkpoint must enable cells to carry out efficient lesion bypass. However, it still remains unclear as to which of the lesion bypass processes depends upon the activation of replication checkpoint. Toward this end, in a previous study we provided evidence that TLS occurs normally in UV-irradiated nucleotide excision repair (NER)-proficient wild-type yeast cells lacking Mec1 or the components of checkpoint clamp and clamp loader (18
). However, when the lesion load becomes greatly accentuated, as in UV-irradiated NER-defective cells, Mec1-mediated phosphorylation of Rev1 contributes to increasing the proficiency of Polζ function in lesion bypass (18
). In addition to the requirement of Mec1/Ddc2 kinase, Rev1 phosphorylation requires the components of checkpoint clamp (Mec3, Rad17, and Ddc1) and clamp loader (Rad24), but Rad53 is not required (18
). Since the frequency of UV-induced mutations shows a reduction in NER-defective yeast cells in combination with the phosphorylation-defectiveness rev1
mutation, the mec1
Δ mutation, or the mec3
Δ, or rad24
Δ mutation and since an epistatic relationship is observed when the rev1
phosphorylation-defectiveness mutation is combined with deletion mutations of these checkpoint proteins, we concluded that Rev1 phosphorylation, mediated by the Mec1/Ddc2 kinase and other checkpoint proteins, contributes to increasing the efficiency of Polζ-dependent TLS, the need for which becomes more acute in NER-defective yeast cells (18
). Overall, these studies have supported the inference that in UV-damaged yeast cells, Polη and Polζ can carry out TLS without the need for Mec1/Rad53-initiated fork stabilization.
Since template switching provides an alternative to TLS, in this study we determine whether replication checkpoint is required for promoting lesion bypass by template switching. We provide evidence that Mec1 and Rad53 are both required for the restoration of normal size to newly synthesized DNA in UV-irradiated yeast cells and suggest that lesion bypass by template switching occurs in conjunction with the stalled replication fork that is maintained at the lesion site by the action of Mec1/Rad53.