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Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5' recessed DNA.

Ellison V, Stillman B - PLoS Biol. (2003)

Bottom Line: RSR preferred DNA substrates possessing 5' recessed ends whereas RFC preferred 3' recessed end DNA substrates.Characterization of the biochemical loading reaction executed by the checkpoint clamp loader RSR suggests new insights into the mechanisms underlying recognition of damage-induced DNA structures and signaling to cell cycle controls.The observation that RSR loads its clamp onto a 5' recessed end supports a potential role for RHR and RSR in diverse DNA metabolism, such as stalled DNA replication forks, recombination-linked DNA repair, and telomere maintenance, among other processes.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.

ABSTRACT
The cellular pathways involved in maintaining genome stability halt cell cycle progression in the presence of DNA damage or incomplete replication. Proteins required for this pathway include Rad17, Rad9, Hus1, Rad1, and Rfc-2, Rfc-3, Rfc-4, and Rfc-5. The heteropentamer replication factor C (RFC) loads during DNA replication the homotrimer proliferating cell nuclear antigen (PCNA) polymerase clamp onto DNA. Sequence similarities suggest the biochemical functions of an RSR (Rad17-Rfc2-Rfc3-Rfc4-Rfc5) complex and an RHR heterotrimer (Rad1-Hus1-Rad9) may be similar to that of RFC and PCNA, respectively. RSR purified from human cells loads RHR onto DNA in an ATP-, replication protein A-, and DNA structure-dependent manner. Interestingly, RSR and RFC differed in their ATPase activities and displayed distinct DNA substrate specificities. RSR preferred DNA substrates possessing 5' recessed ends whereas RFC preferred 3' recessed end DNA substrates. Characterization of the biochemical loading reaction executed by the checkpoint clamp loader RSR suggests new insights into the mechanisms underlying recognition of damage-induced DNA structures and signaling to cell cycle controls. The observation that RSR loads its clamp onto a 5' recessed end supports a potential role for RHR and RSR in diverse DNA metabolism, such as stalled DNA replication forks, recombination-linked DNA repair, and telomere maintenance, among other processes.

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Possible Substrates onto Which the Checkpoint Clamp Loader RSR May Load Its Clamp (RHR)DNA maintenance pathways, including those depicted here, generate intermediates containing free and/or recessed 3′ ends that are processed by a variety of proteins. These structures also contain recessed 5′ ends, whose fate in these reactions is unclear. Given that RSR loads RHR (depicted as a ring or donut encircling the DNA) onto recessed 5′ ends in vitro, recessed 5′ ends generated in vivo in the depicted pathways can be considered potential substrates. They all contain adjacent single-stranded DNA that could be bound by RPA. RHR has been shown to be required for checkpoint signaling in response to DNA replication fork arrest (Longhese et al. 1997), double-strand breaks (Kondo et al. 2001; Melo et al. 2001), and improper telomere maintenance (Garvik et al. 1995; Lydall and Weinert 1995; Longhese et al. 2000). The RHR clamp is proposed to protect the recessed 5′ end from extensive degradation by exonucleases and to promote resolution of these structures back to duplex DNA.
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pbio.0000033-g007: Possible Substrates onto Which the Checkpoint Clamp Loader RSR May Load Its Clamp (RHR)DNA maintenance pathways, including those depicted here, generate intermediates containing free and/or recessed 3′ ends that are processed by a variety of proteins. These structures also contain recessed 5′ ends, whose fate in these reactions is unclear. Given that RSR loads RHR (depicted as a ring or donut encircling the DNA) onto recessed 5′ ends in vitro, recessed 5′ ends generated in vivo in the depicted pathways can be considered potential substrates. They all contain adjacent single-stranded DNA that could be bound by RPA. RHR has been shown to be required for checkpoint signaling in response to DNA replication fork arrest (Longhese et al. 1997), double-strand breaks (Kondo et al. 2001; Melo et al. 2001), and improper telomere maintenance (Garvik et al. 1995; Lydall and Weinert 1995; Longhese et al. 2000). The RHR clamp is proposed to protect the recessed 5′ end from extensive degradation by exonucleases and to promote resolution of these structures back to duplex DNA.

Mentions: Based on the aforementioned observations and findings that the human and S. pombe rad17 proteins were reported to bind chromatin constitutively (Kai et al. 2001), it was suggested that the RHR clamp and the RSR clamp loader may function as initial sensors of DNA damage. This model, however, is inconsistent with our findings revealing a requirement for a 5′ recessed primer–template substrate and RPA for loading of the RHR complex by the RSR clamp loader. RPA, an essential component of DNA replication, repair, and recombination pathways in vivo (Longhese et al. 1994; Wold 1997), has been demonstrated to be a substrate for kinases involved in the DNA damage response pathway (Brush and Kelly 2000; Oakley et al. 2001). S. cerevisiae RPA has been shown to genetically interact with Rfc4, and allele-specific mutants in RPA that abolish the interaction with RFC in vitro have been shown to confer a DNA damage checkpoint-deficient phenotype in vivo (Kim and Brill 2001). In addition, activation of the S-phase DNA damage checkpoint in a Xenopus cell-free DNA replication system resulted in RPA- and polα/primase-dependent loading of Xenopus Rad17 and Hus1 proteins onto damaged chromatin (You et al. 2002; Lee et al. 2003). Therefore, we suggest a model in which the checkpoint clamp and clamp loader function not as initial sensors of DNA damage, but instead play a vital role in DNA damage responses by stabilizing stalled replication forks and/or stimulating replication fork reactivation and recombination-dependent DNA replication pathways after lesions are first processed into structures that are suitable substrates for clamp loading (Figure 7). All of these structures have 5′ recessed ends and RPA could bind to the single-stranded DNA. We further suggest that a modified version of RPA would be primarily responsible for recruiting the checkpoint clamp loader and the clamp to these sites. Under these circumstances, essential roles of the checkpoint clamp may be to protect 5′ recessed ends from exonucleolytic degradation and promote resolution of these abnormal structures in DNA.


Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5' recessed DNA.

Ellison V, Stillman B - PLoS Biol. (2003)

Possible Substrates onto Which the Checkpoint Clamp Loader RSR May Load Its Clamp (RHR)DNA maintenance pathways, including those depicted here, generate intermediates containing free and/or recessed 3′ ends that are processed by a variety of proteins. These structures also contain recessed 5′ ends, whose fate in these reactions is unclear. Given that RSR loads RHR (depicted as a ring or donut encircling the DNA) onto recessed 5′ ends in vitro, recessed 5′ ends generated in vivo in the depicted pathways can be considered potential substrates. They all contain adjacent single-stranded DNA that could be bound by RPA. RHR has been shown to be required for checkpoint signaling in response to DNA replication fork arrest (Longhese et al. 1997), double-strand breaks (Kondo et al. 2001; Melo et al. 2001), and improper telomere maintenance (Garvik et al. 1995; Lydall and Weinert 1995; Longhese et al. 2000). The RHR clamp is proposed to protect the recessed 5′ end from extensive degradation by exonucleases and to promote resolution of these structures back to duplex DNA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC261875&req=5

pbio.0000033-g007: Possible Substrates onto Which the Checkpoint Clamp Loader RSR May Load Its Clamp (RHR)DNA maintenance pathways, including those depicted here, generate intermediates containing free and/or recessed 3′ ends that are processed by a variety of proteins. These structures also contain recessed 5′ ends, whose fate in these reactions is unclear. Given that RSR loads RHR (depicted as a ring or donut encircling the DNA) onto recessed 5′ ends in vitro, recessed 5′ ends generated in vivo in the depicted pathways can be considered potential substrates. They all contain adjacent single-stranded DNA that could be bound by RPA. RHR has been shown to be required for checkpoint signaling in response to DNA replication fork arrest (Longhese et al. 1997), double-strand breaks (Kondo et al. 2001; Melo et al. 2001), and improper telomere maintenance (Garvik et al. 1995; Lydall and Weinert 1995; Longhese et al. 2000). The RHR clamp is proposed to protect the recessed 5′ end from extensive degradation by exonucleases and to promote resolution of these structures back to duplex DNA.
Mentions: Based on the aforementioned observations and findings that the human and S. pombe rad17 proteins were reported to bind chromatin constitutively (Kai et al. 2001), it was suggested that the RHR clamp and the RSR clamp loader may function as initial sensors of DNA damage. This model, however, is inconsistent with our findings revealing a requirement for a 5′ recessed primer–template substrate and RPA for loading of the RHR complex by the RSR clamp loader. RPA, an essential component of DNA replication, repair, and recombination pathways in vivo (Longhese et al. 1994; Wold 1997), has been demonstrated to be a substrate for kinases involved in the DNA damage response pathway (Brush and Kelly 2000; Oakley et al. 2001). S. cerevisiae RPA has been shown to genetically interact with Rfc4, and allele-specific mutants in RPA that abolish the interaction with RFC in vitro have been shown to confer a DNA damage checkpoint-deficient phenotype in vivo (Kim and Brill 2001). In addition, activation of the S-phase DNA damage checkpoint in a Xenopus cell-free DNA replication system resulted in RPA- and polα/primase-dependent loading of Xenopus Rad17 and Hus1 proteins onto damaged chromatin (You et al. 2002; Lee et al. 2003). Therefore, we suggest a model in which the checkpoint clamp and clamp loader function not as initial sensors of DNA damage, but instead play a vital role in DNA damage responses by stabilizing stalled replication forks and/or stimulating replication fork reactivation and recombination-dependent DNA replication pathways after lesions are first processed into structures that are suitable substrates for clamp loading (Figure 7). All of these structures have 5′ recessed ends and RPA could bind to the single-stranded DNA. We further suggest that a modified version of RPA would be primarily responsible for recruiting the checkpoint clamp loader and the clamp to these sites. Under these circumstances, essential roles of the checkpoint clamp may be to protect 5′ recessed ends from exonucleolytic degradation and promote resolution of these abnormal structures in DNA.

Bottom Line: RSR preferred DNA substrates possessing 5' recessed ends whereas RFC preferred 3' recessed end DNA substrates.Characterization of the biochemical loading reaction executed by the checkpoint clamp loader RSR suggests new insights into the mechanisms underlying recognition of damage-induced DNA structures and signaling to cell cycle controls.The observation that RSR loads its clamp onto a 5' recessed end supports a potential role for RHR and RSR in diverse DNA metabolism, such as stalled DNA replication forks, recombination-linked DNA repair, and telomere maintenance, among other processes.

View Article: PubMed Central - PubMed

Affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.

ABSTRACT
The cellular pathways involved in maintaining genome stability halt cell cycle progression in the presence of DNA damage or incomplete replication. Proteins required for this pathway include Rad17, Rad9, Hus1, Rad1, and Rfc-2, Rfc-3, Rfc-4, and Rfc-5. The heteropentamer replication factor C (RFC) loads during DNA replication the homotrimer proliferating cell nuclear antigen (PCNA) polymerase clamp onto DNA. Sequence similarities suggest the biochemical functions of an RSR (Rad17-Rfc2-Rfc3-Rfc4-Rfc5) complex and an RHR heterotrimer (Rad1-Hus1-Rad9) may be similar to that of RFC and PCNA, respectively. RSR purified from human cells loads RHR onto DNA in an ATP-, replication protein A-, and DNA structure-dependent manner. Interestingly, RSR and RFC differed in their ATPase activities and displayed distinct DNA substrate specificities. RSR preferred DNA substrates possessing 5' recessed ends whereas RFC preferred 3' recessed end DNA substrates. Characterization of the biochemical loading reaction executed by the checkpoint clamp loader RSR suggests new insights into the mechanisms underlying recognition of damage-induced DNA structures and signaling to cell cycle controls. The observation that RSR loads its clamp onto a 5' recessed end supports a potential role for RHR and RSR in diverse DNA metabolism, such as stalled DNA replication forks, recombination-linked DNA repair, and telomere maintenance, among other processes.

Show MeSH
Related in: MedlinePlus