Requirement of replication checkpoint protein kinases Mec1/Rad53 for postreplication repair in yeast

Abstract

DNA lesions in the template strand block the replication fork. In Saccharomyces cerevisiae, replication through DNA lesions occurs via a Rad6/Rad18-dependent pathway where lesions can be bypassed by the action of translesion synthesis (TLS) DNA polymerases η and ζ or by Rad5-mediated template switching. An alternative Rad6/Rad18-independent but Rad52-dependent template switching pathway can also restore the continuity of the replication fork. The Mec1/Rad53-dependent replication checkpoint plays a crucial role in the maintenance of stable and functional replication forks in yeast cells with DNA damage; however, it has remained unclear which of the lesion bypass processes requires the activation of replication checkpoint-mediated fork stabilization. Here we show that postreplication repair (PRR) of newly synthesized DNA in UV-damaged yeast cells is inhibited in the absence of Mec1 and Rad53 proteins. Since TLS remains functional in cells lacking these checkpoint kinases and since template switching by the Rad5 and Rad52 pathways provides the alternative means of lesion bypass and requires Mec1/Rad53, we infer that lesion bypass by the template switching pathways occurs in conjunction with the replication fork that has been stabilized at the lesion site by the action of Mec1/Rad53-mediated replication checkpoint.

Importance: Eukaryotic cells possess mechanisms called checkpoints that act to stop the cell cycle when DNA replication is halted by lesions in the template strand. Upon stalling of the ongoing replication at the lesion site, the recruitment of Mec1 and Rad53 kinases to the replication ensemble initiates the checkpoint wherein Mec1-mediated phosphorylation of Rad53 activates the pathway. A crucial role of replication checkpoint is to stabilize the replication fork by maintaining the association of DNA polymerases with the other replication components at the stall site. Our observations that Mec1 and Rad53 are required for lesion bypass by template switching have important implications for whether lesion bypass occurs in conjunction with the stalled replication ensemble or in gaps that could have been left behind the newly restarted forks. We discuss this important issue and suggest that lesion bypass in Saccharomyces cerevisiae cells occurs in conjunction with the stalled replication forks and not in gaps.

FIG 1

Synergistic enhancement of UV sensitivity of rad14Δ cells in the absence of Mec1 and Rad53. Survival after UV irradiation of wild-type strain EMY74.7 and its isogenic derivative strains: sml1Δ mec1Δ strain (A), sml1Δ rad53Δ strain (B), sml1Δ mec1Δ rad14Δ strain (C), and sml1Δ rad53Δ rad14Δ strain (D). Survival curves represent averages of at least three different experiments for each strain. Error bars represent standard deviations of determinations. The apparent absence of error bars in some cases is because the error bars are very small.

FIG 2

The absence of Mec1 or Rad53 confers only a modest increase in the UV sensitivity of mutants defective in lesion bypass by template switching. Survival after UV irradiation of isogenic derivative strains of EMY74.7: sml1Δ mec1Δ rad18Δ strain (A), sml1Δ rad53Δ rad18Δ strain (B), sml1Δ mec1Δ rad5Δ strain (C), sml1Δ rad53Δ rad5Δ strain (D), sml1Δ mec1Δ rad51Δ strain (E), and sml1Δ rad53Δ rad51Δ strain (F). Survival curves represent averages of at least three different experiments for each strain. Error bars represent standard deviations of determinations. The absence of error bars in some cases is because the error bars are very small.

FIG 3

Requirement of Mec1 and Rad53 for postreplication repair of UV-damaged DNA. Sedimentation in alkaline sucrose gradients of nuclear DNA from cells incubated for different periods following UV irradiation with 3.5 J/m². sml1Δ rad1Δ (A), sml1Δ mec1Δ rad1Δ (B), and sml1Δ rad53Δ rad1Δ (C) strains, respectively, were UV irradiated at 3.5 J/m² and then pulse-labeled with [³H]uracil for 15 min, followed by different periods to allow for repair in high-uracil medium: 30 min (Δ), 2 h (□), and 4 h (●) (A) and 30 min (Δ) and 6 h (●) (B and C). DNA from unirradiated cells was pulse-labeled with [³H]uracil for 15 min followed by incubation for 6 h (○); a similar sedimentation pattern was attained in unirradiated cells pulse-labeled for 15 min following by a chase for 30 min in high-uracil medium (data not shown).

FIG 4

Lesion bypass by translesion synthesis remains operational in the absence of Mec1 and Rad53. (A and B) UV survival (left) and frequencies of UV-induced can1^r mutations (right) were determined for the sml1Δ rad53Δ strain in combination with the rev3Δ or rad30Δ mutation. (C) UV survival of sml1Δ mec1Δ rad14Δ (left) and sml1Δ rad53Δ rad14Δ (right) strains in combination with the rad30Δ mutation. Survival curves represent averages of at least three different experiments for each strain. Error bars represent standard deviations of determinations. The absence of error bars in some cases is because the error bars are very small.

FIG 5

Inactivation of translesion synthesis leads to further impairment of residual postreplication repair that occurs in the absence of replication checkpoint. Sedimentation in alkaline sucrose gradients of nuclear DNA from cells incubated for different periods following UV irradiation with 3.5 J/m². sml1Δ mec1Δ rad1Δ rad30Δ (A), sml1Δ rad53Δ rad1Δ rad30Δ (B), sml1Δ mec1Δ rad1Δ rad30Δ rev3Δ (C), and sml1Δ rad53Δ rad1Δ rad30Δ rev3Δ (D) strains, respectively, were UV irradiated at 3.5 J/m² and then pulse-labeled with [³H]uracil for 15 min, followed by a 30-min chase (Δ) or a 6-h repair period (●) in high-uracil medium. Also shown is the sedimentation pattern of DNA from unirradiated cells pulse-labeled with [³H]uracil for 15 min, followed by a 6-h incubation (○).

FIG 6

Model for role of Mec1- and Rad53-mediated replication checkpoint in lesion bypass. It is proposed that lesion bypass by TLS or by template switching occurs in coordination with the replication fork and not in gaps that might have been left behind opposite from DNA lesions and then filled in later by these lesion bypass processes during the G₂ phase. Since Mec1 and Rad53 are required for postreplication repair of UV-damaged DNA but TLS remains functional in the absence of these replication checkpoint proteins, we posit that both the Rad6-Rad18-Rad5-dependent and the Rad51-Rad52-Rad54-dependent template switching pathways require the Mec1/Rad53-mediated fork stabilization. From the observations that PCNA ubiquitylation is restricted primarily to S phase in UV-irradiated yeast cells (27) and that TLS remains functional in the absence of Mec1 and Rad53 (18), we infer that TLS occurs in coordination with the replication fork, but that does not necessitate the imposition of replication checkpoint. Presumably, TLS can occur in the absence of checkpoint, perhaps because of its being a less cumbersome and more efficient process than template switching.