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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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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.
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f6: 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.

Mentions: The suggested requirement of Mec1/Rad53-mediated fork stabilization for promoting lesion bypass by template switching raises the question of why a stable fork has to be maintained during this process but is not required for TLS. As we have suggested before (18), the lack of requirement of replication checkpoint for TLS could be explained if we assume that TLS occurs in a relatively rapid manner and that because of the rapidity of lesion bypass, the short duration of the fork remaining stalled does not generate enough of a signal necessary for checkpoint activation to occur. In contrast, since lesion bypass by the Rad5 and Rad51/Rad52 pathways involving template switching and copy-choice type of DNA synthesis is likely to be a much more intricate process than TLS, involving a number of discrete reactions, we expect that the fork remains stalled for a period sufficient for checkpoint activation to occur. Further, the prolonged stabilization of the replication fork needed for template switching could be maintained only if the Mec1/Rad53-initiated checkpoint were installed. A summary of these ideas is presented in Fig. 6.


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

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

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

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

f6: 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.
Mentions: The suggested requirement of Mec1/Rad53-mediated fork stabilization for promoting lesion bypass by template switching raises the question of why a stable fork has to be maintained during this process but is not required for TLS. As we have suggested before (18), the lack of requirement of replication checkpoint for TLS could be explained if we assume that TLS occurs in a relatively rapid manner and that because of the rapidity of lesion bypass, the short duration of the fork remaining stalled does not generate enough of a signal necessary for checkpoint activation to occur. In contrast, since lesion bypass by the Rad5 and Rad51/Rad52 pathways involving template switching and copy-choice type of DNA synthesis is likely to be a much more intricate process than TLS, involving a number of discrete reactions, we expect that the fork remains stalled for a period sufficient for checkpoint activation to occur. Further, the prolonged stabilization of the replication fork needed for template switching could be maintained only if the Mec1/Rad53-initiated checkpoint were installed. A summary of these ideas is presented in Fig. 6.

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