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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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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).
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f3: 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).

Mentions: For PRR studies, the NER-defective rad1Δ mutant was combined with the sml1Δ mutation, and following UV irradiation of sml1Δ rad1Δ cells at 3.5 J/m2, the size of newly synthesized DNA from UV-damaged templates was examined by pulse-labeling of DNA with [3H]uracil for 15 min followed by a chase period of 30 min in high-uracil medium. Following this treatment, DNA sediments toward the top of the alkaline sucrose gradient (Fig. 3A); this small size of DNA results from the presence of discontinuities in the newly synthesized strand that arise from the stalling of replication forks opposite from lesion sites. In contrast, in unirradiated sml1Δ rad1Δ cells, the newly synthesized DNA attains normal size following such a 15-min pulse and 30-min chase in high-uracil medium (data not shown). In sml1Δ rad1Δ cells that were UV irradiated and where, following the 15-min pulse-labeling period, cells were allowed to repair the discontinuities for 2 h, the newly synthesized DNA attained almost the same size as that in unirradiated control cells, whereas a 4-h repair period following the 15-min pulse restored normal size to newly synthesized DNA (Fig. 3A).


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

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

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

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

f3: 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).
Mentions: For PRR studies, the NER-defective rad1Δ mutant was combined with the sml1Δ mutation, and following UV irradiation of sml1Δ rad1Δ cells at 3.5 J/m2, the size of newly synthesized DNA from UV-damaged templates was examined by pulse-labeling of DNA with [3H]uracil for 15 min followed by a chase period of 30 min in high-uracil medium. Following this treatment, DNA sediments toward the top of the alkaline sucrose gradient (Fig. 3A); this small size of DNA results from the presence of discontinuities in the newly synthesized strand that arise from the stalling of replication forks opposite from lesion sites. In contrast, in unirradiated sml1Δ rad1Δ cells, the newly synthesized DNA attains normal size following such a 15-min pulse and 30-min chase in high-uracil medium (data not shown). In sml1Δ rad1Δ cells that were UV irradiated and where, following the 15-min pulse-labeling period, cells were allowed to repair the discontinuities for 2 h, the newly synthesized DNA attained almost the same size as that in unirradiated control cells, whereas a 4-h repair period following the 15-min pulse restored normal size to newly synthesized DNA (Fig. 3A).

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