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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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RHR Forms a Sliding Clamp on DNALoading reactions in the experiments represented in (A) and (B) were performed as described in the Materials and Methods using either recessed 5′ or recessed 3′ primer–template DNA–RPA substrates bound to beads, and then recovery of proteins with the beads was analyzed by Wb for the indicated proteins. In each experiment using template strands biotinylated at either the 5′ or 3′ end, either both ends of the DNA substrate (reactions represented in lanes 2–5) or only one end of the substrate (reactions represented in lanes 6–9) was blocked by selectively positioning the bead relative to RPA—either at the opposite end of the DNA (distal, therefore both ends blocked) or at the same end of the DNA (proximal, therefore only one end blocked). In (A), lanes represent reactions that contained 0.25 pmol of RFC (except for those in lanes 2 and 6), 2 pmol of PCNA, and either a 3′ recessed primer/3′ biotin template DNA (bead distal to RPA; lanes 2–5), a 3′ recessed primer/5′ biotin template DNA (bead proximal to RPA; lanes 6–9), or no DNA (lane 1). In (B), lanes represent reactions that contained 0.25 pmol of RSR (except for those in lanes 2 and 6), 1 pmol of RHR, and either a 5′ recessed primer/5′ biotin template (bead distal to RPA; lanes 2–5), a 5′ recessed/3′ biotin template (bead proximal to RPA; lanes 6–9) or no DNA (lane 1). In both (A) and (B), reactions were performed in the absence (lanes 3 and 7) or presence of nucleotide (ATP: lanes 2, 4, 6, and 8; ATPγS: lanes 5 and 9), and 20% of the reaction input was loaded in the lane labeled L.
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pbio.0000033-g005: RHR Forms a Sliding Clamp on DNALoading reactions in the experiments represented in (A) and (B) were performed as described in the Materials and Methods using either recessed 5′ or recessed 3′ primer–template DNA–RPA substrates bound to beads, and then recovery of proteins with the beads was analyzed by Wb for the indicated proteins. In each experiment using template strands biotinylated at either the 5′ or 3′ end, either both ends of the DNA substrate (reactions represented in lanes 2–5) or only one end of the substrate (reactions represented in lanes 6–9) was blocked by selectively positioning the bead relative to RPA—either at the opposite end of the DNA (distal, therefore both ends blocked) or at the same end of the DNA (proximal, therefore only one end blocked). In (A), lanes represent reactions that contained 0.25 pmol of RFC (except for those in lanes 2 and 6), 2 pmol of PCNA, and either a 3′ recessed primer/3′ biotin template DNA (bead distal to RPA; lanes 2–5), a 3′ recessed primer/5′ biotin template DNA (bead proximal to RPA; lanes 6–9), or no DNA (lane 1). In (B), lanes represent reactions that contained 0.25 pmol of RSR (except for those in lanes 2 and 6), 1 pmol of RHR, and either a 5′ recessed primer/5′ biotin template (bead distal to RPA; lanes 2–5), a 5′ recessed/3′ biotin template (bead proximal to RPA; lanes 6–9) or no DNA (lane 1). In both (A) and (B), reactions were performed in the absence (lanes 3 and 7) or presence of nucleotide (ATP: lanes 2, 4, 6, and 8; ATPγS: lanes 5 and 9), and 20% of the reaction input was loaded in the lane labeled L.

Mentions: To test whether PCNA was topologically linked in this assay, we compared loading onto recessed 3′ end primer–template DNAs that contained the biotin-linked bead at either the double-stranded end (bead distal to RPA) or at the single-stranded end of the template (bead proximal to RPA) (Figure 5A). In contrast to the former substrate, the latter possesses one unblocked or “free” end that would allow the PCNA to slide off the duplex DNA. Thus, if the PCNA was topologically linked to the DNA, very poor recovery of the clamp on this substrate was expected. As predicted, although efficiently recovered on the DNA with the biotin bead complex distal to RPA (Figure 5A, lane 4; see also the experiment presented in Figure 4), PCNA was not recovered when the biotin bead complex was placed at the single-stranded end of the template (proximal to RPA) (Figure 5A, lane 8). Placement of the biotin at the single-stranded end of the DNA did not simply inhibit RFC activity, for ternary complex formation on both substrates in the presence of ATPγS was observed with similar efficiencies (Figure 5A, lanes 5 and 9). In addition, both the 5′ biotin and 3′ biotin substrates were capable of activating the ATPase activity of RFC with the same efficiency (data not shown), again indicating no specific impairment of RFC function by the 5′ biotin substrate. We concluded, therefore, that the inability to recover the PCNA by unblocking one end of the substrate was due to PCNA sliding off the duplex DNA after it was loaded. Thus, in this assay, the PCNA was topologically linked to the 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)

RHR Forms a Sliding Clamp on DNALoading reactions in the experiments represented in (A) and (B) were performed as described in the Materials and Methods using either recessed 5′ or recessed 3′ primer–template DNA–RPA substrates bound to beads, and then recovery of proteins with the beads was analyzed by Wb for the indicated proteins. In each experiment using template strands biotinylated at either the 5′ or 3′ end, either both ends of the DNA substrate (reactions represented in lanes 2–5) or only one end of the substrate (reactions represented in lanes 6–9) was blocked by selectively positioning the bead relative to RPA—either at the opposite end of the DNA (distal, therefore both ends blocked) or at the same end of the DNA (proximal, therefore only one end blocked). In (A), lanes represent reactions that contained 0.25 pmol of RFC (except for those in lanes 2 and 6), 2 pmol of PCNA, and either a 3′ recessed primer/3′ biotin template DNA (bead distal to RPA; lanes 2–5), a 3′ recessed primer/5′ biotin template DNA (bead proximal to RPA; lanes 6–9), or no DNA (lane 1). In (B), lanes represent reactions that contained 0.25 pmol of RSR (except for those in lanes 2 and 6), 1 pmol of RHR, and either a 5′ recessed primer/5′ biotin template (bead distal to RPA; lanes 2–5), a 5′ recessed/3′ biotin template (bead proximal to RPA; lanes 6–9) or no DNA (lane 1). In both (A) and (B), reactions were performed in the absence (lanes 3 and 7) or presence of nucleotide (ATP: lanes 2, 4, 6, and 8; ATPγS: lanes 5 and 9), and 20% of the reaction input was loaded in the lane labeled L.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC261875&req=5

pbio.0000033-g005: RHR Forms a Sliding Clamp on DNALoading reactions in the experiments represented in (A) and (B) were performed as described in the Materials and Methods using either recessed 5′ or recessed 3′ primer–template DNA–RPA substrates bound to beads, and then recovery of proteins with the beads was analyzed by Wb for the indicated proteins. In each experiment using template strands biotinylated at either the 5′ or 3′ end, either both ends of the DNA substrate (reactions represented in lanes 2–5) or only one end of the substrate (reactions represented in lanes 6–9) was blocked by selectively positioning the bead relative to RPA—either at the opposite end of the DNA (distal, therefore both ends blocked) or at the same end of the DNA (proximal, therefore only one end blocked). In (A), lanes represent reactions that contained 0.25 pmol of RFC (except for those in lanes 2 and 6), 2 pmol of PCNA, and either a 3′ recessed primer/3′ biotin template DNA (bead distal to RPA; lanes 2–5), a 3′ recessed primer/5′ biotin template DNA (bead proximal to RPA; lanes 6–9), or no DNA (lane 1). In (B), lanes represent reactions that contained 0.25 pmol of RSR (except for those in lanes 2 and 6), 1 pmol of RHR, and either a 5′ recessed primer/5′ biotin template (bead distal to RPA; lanes 2–5), a 5′ recessed/3′ biotin template (bead proximal to RPA; lanes 6–9) or no DNA (lane 1). In both (A) and (B), reactions were performed in the absence (lanes 3 and 7) or presence of nucleotide (ATP: lanes 2, 4, 6, and 8; ATPγS: lanes 5 and 9), and 20% of the reaction input was loaded in the lane labeled L.
Mentions: To test whether PCNA was topologically linked in this assay, we compared loading onto recessed 3′ end primer–template DNAs that contained the biotin-linked bead at either the double-stranded end (bead distal to RPA) or at the single-stranded end of the template (bead proximal to RPA) (Figure 5A). In contrast to the former substrate, the latter possesses one unblocked or “free” end that would allow the PCNA to slide off the duplex DNA. Thus, if the PCNA was topologically linked to the DNA, very poor recovery of the clamp on this substrate was expected. As predicted, although efficiently recovered on the DNA with the biotin bead complex distal to RPA (Figure 5A, lane 4; see also the experiment presented in Figure 4), PCNA was not recovered when the biotin bead complex was placed at the single-stranded end of the template (proximal to RPA) (Figure 5A, lane 8). Placement of the biotin at the single-stranded end of the DNA did not simply inhibit RFC activity, for ternary complex formation on both substrates in the presence of ATPγS was observed with similar efficiencies (Figure 5A, lanes 5 and 9). In addition, both the 5′ biotin and 3′ biotin substrates were capable of activating the ATPase activity of RFC with the same efficiency (data not shown), again indicating no specific impairment of RFC function by the 5′ biotin substrate. We concluded, therefore, that the inability to recover the PCNA by unblocking one end of the substrate was due to PCNA sliding off the duplex DNA after it was loaded. Thus, in this assay, the PCNA was topologically linked to the 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