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

Gangavarapu V, Santa Maria SR, Prakash S, Prakash L - MBio (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, Galveston, Texas, USA.

ABSTRACT

Unlabelled: 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.

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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 (○).
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f5: 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 (○).

Mentions: Since TLS remains functional in the absence of Mec1/Rad53 kinases, we determined whether the residual repair of discontinuities in the newly synthesized DNA that occurs in UV-irradiated sml1Δ rad1Δ mec1Δ and sml1Δ rad1Δ rad53Δ cells derives from the TLS role of Polη and Polζ. To verify this possibility, we examined the size of newly synthesized DNA in sml1Δ rad1Δ mec1Δ rad30Δ and sml1Δ rad1Δ rad53Δ rad30Δ strains following UV irradiation at 3.5 J/m2. A comparison of gradient profiles in Fig. 5A and B with those in Fig. 3B and C, respectively, shows that in the absence of Polη, DNA synthesized from UV-damaged templates does not attain as large a size as it does in the presence of Polη. A much greater reduction in the capacity to repair discontinuities in the newly synthesized DNA is observed in the sml1Δ rad1Δ mec1Δ rad30Δ rev3Δ and sml1Δ rad1Δ rad53Δ rad30Δ rev3Δ strains such that the gradient profiles of DNA from UV-irradiated cells pulse-labeled for 15 min followed by a chase period of 30 min or a repair period of 6 h become more coincident (Fig. 5C and D). We conclude from these observations that Polη and Polζ are able to function in lesion bypass in the absence of Mec1 and Rad53.


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

Gangavarapu V, Santa Maria SR, Prakash S, Prakash L - MBio (2011)

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 (○).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101783&req=5

f5: 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 (○).
Mentions: Since TLS remains functional in the absence of Mec1/Rad53 kinases, we determined whether the residual repair of discontinuities in the newly synthesized DNA that occurs in UV-irradiated sml1Δ rad1Δ mec1Δ and sml1Δ rad1Δ rad53Δ cells derives from the TLS role of Polη and Polζ. To verify this possibility, we examined the size of newly synthesized DNA in sml1Δ rad1Δ mec1Δ rad30Δ and sml1Δ rad1Δ rad53Δ rad30Δ strains following UV irradiation at 3.5 J/m2. A comparison of gradient profiles in Fig. 5A and B with those in Fig. 3B and C, respectively, shows that in the absence of Polη, DNA synthesized from UV-damaged templates does not attain as large a size as it does in the presence of Polη. A much greater reduction in the capacity to repair discontinuities in the newly synthesized DNA is observed in the sml1Δ rad1Δ mec1Δ rad30Δ rev3Δ and sml1Δ rad1Δ rad53Δ rad30Δ rev3Δ strains such that the gradient profiles of DNA from UV-irradiated cells pulse-labeled for 15 min followed by a chase period of 30 min or a repair period of 6 h become more coincident (Fig. 5C and D). We conclude from these observations that Polη and Polζ are able to function in lesion bypass in the absence of Mec1 and Rad53.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, Galveston, Texas, USA.

ABSTRACT

Unlabelled: 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.

Show MeSH
Related in: MedlinePlus