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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 Loading Is Nucleotide, Primer, and RPA DependentLoading reactions represented in (A) were performed with 2 pmol of PCNA, 0.25 pmol of RFC, and the 5′ and 3′ recessed primer–template DNA–RPA complex bound to beads, whereas those in (B) were performed with 1 pmol of RHR, 0.25 pmol of RSR, and the 5′ and 3′ recessed primer–template DNA–RPA complex bound to beads. Reactions were performed as described in the Materials and Methods, and the bead-precipitated proteins were then analyzed by Wb for PCNA, RFC, Rad17, Rad9, Hus1, and Rad1, respectively. In both (A) and (B), lanes 2 and 9 represent reactions that contained the clamp alone (PCNA in [A], RHR in [B]), and all reactions represented in lanes 1, 3–8, and 10–12, contained both the clamp and its corresponding clamp loader. In both (A) and (B), reactions were performed in the absence of nucleotide (lanes 3, 6, and 9) or in the presence of ATP (lanes 1, 2, 4, 7, 9, and 11) or ATPγS (lanes 5, 8, and 12). All reactions contained RPA except those in lanes 6–8, and all reactions contained primer–template DNA bound beads except that in lane 1 (beads without DNA) and those in lanes 6–9 (template DNA alone bound beads).
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pbio.0000033-g003: RHR Loading Is Nucleotide, Primer, and RPA DependentLoading reactions represented in (A) were performed with 2 pmol of PCNA, 0.25 pmol of RFC, and the 5′ and 3′ recessed primer–template DNA–RPA complex bound to beads, whereas those in (B) were performed with 1 pmol of RHR, 0.25 pmol of RSR, and the 5′ and 3′ recessed primer–template DNA–RPA complex bound to beads. Reactions were performed as described in the Materials and Methods, and the bead-precipitated proteins were then analyzed by Wb for PCNA, RFC, Rad17, Rad9, Hus1, and Rad1, respectively. In both (A) and (B), lanes 2 and 9 represent reactions that contained the clamp alone (PCNA in [A], RHR in [B]), and all reactions represented in lanes 1, 3–8, and 10–12, contained both the clamp and its corresponding clamp loader. In both (A) and (B), reactions were performed in the absence of nucleotide (lanes 3, 6, and 9) or in the presence of ATP (lanes 1, 2, 4, 7, 9, and 11) or ATPγS (lanes 5, 8, and 12). All reactions contained RPA except those in lanes 6–8, and all reactions contained primer–template DNA bound beads except that in lane 1 (beads without DNA) and those in lanes 6–9 (template DNA alone bound beads).

Mentions: Using the same relative amounts of each fraction present in the silver-stained gel (Figure 1A), the affinity-purified RSR was analyzed for RHR loading (Figure 1F). The initial fraction that was loaded on to the Rad17 affinity column was functional, as recovery of RHR with the beads was not observed in reactions that lacked DNA (Figure 1F, lane 1), the Rad17-containing fraction (Figure 1F, lane2), or nucleotide (Figure 1F, lane 3). Consistent with a requirement for Rad17 for RHR loading in vitro, the flowthrough from the affinity column that lacked Rad17 but contained other Rfc2-containing complexes (such as RFC) was inactive (Figure 1F, lanes 6–8). The purified RSR was capable of loading RHR onto DNA in a nucleotide-dependent manner, similar to the starting material (compare Figure 1A, lanes 1 and 7; Figure 1F, lanes 3–5 and 12–14). The four small RFC subunit complex, which by itself does did not bind DNA nor load RHR and PCNA onto DNA in this assay (data not shown), was also detected on DNA when Rad17 was present (Figure 1F; see also Figure 3B), further suggesting that Rfc2–5 functions in a complex with Rad17. Thus, a complex of RSR that was purified from human cells could load the RHR onto DNA in vitro.


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 Loading Is Nucleotide, Primer, and RPA DependentLoading reactions represented in (A) were performed with 2 pmol of PCNA, 0.25 pmol of RFC, and the 5′ and 3′ recessed primer–template DNA–RPA complex bound to beads, whereas those in (B) were performed with 1 pmol of RHR, 0.25 pmol of RSR, and the 5′ and 3′ recessed primer–template DNA–RPA complex bound to beads. Reactions were performed as described in the Materials and Methods, and the bead-precipitated proteins were then analyzed by Wb for PCNA, RFC, Rad17, Rad9, Hus1, and Rad1, respectively. In both (A) and (B), lanes 2 and 9 represent reactions that contained the clamp alone (PCNA in [A], RHR in [B]), and all reactions represented in lanes 1, 3–8, and 10–12, contained both the clamp and its corresponding clamp loader. In both (A) and (B), reactions were performed in the absence of nucleotide (lanes 3, 6, and 9) or in the presence of ATP (lanes 1, 2, 4, 7, 9, and 11) or ATPγS (lanes 5, 8, and 12). All reactions contained RPA except those in lanes 6–8, and all reactions contained primer–template DNA bound beads except that in lane 1 (beads without DNA) and those in lanes 6–9 (template DNA alone bound beads).
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Related In: Results  -  Collection

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pbio.0000033-g003: RHR Loading Is Nucleotide, Primer, and RPA DependentLoading reactions represented in (A) were performed with 2 pmol of PCNA, 0.25 pmol of RFC, and the 5′ and 3′ recessed primer–template DNA–RPA complex bound to beads, whereas those in (B) were performed with 1 pmol of RHR, 0.25 pmol of RSR, and the 5′ and 3′ recessed primer–template DNA–RPA complex bound to beads. Reactions were performed as described in the Materials and Methods, and the bead-precipitated proteins were then analyzed by Wb for PCNA, RFC, Rad17, Rad9, Hus1, and Rad1, respectively. In both (A) and (B), lanes 2 and 9 represent reactions that contained the clamp alone (PCNA in [A], RHR in [B]), and all reactions represented in lanes 1, 3–8, and 10–12, contained both the clamp and its corresponding clamp loader. In both (A) and (B), reactions were performed in the absence of nucleotide (lanes 3, 6, and 9) or in the presence of ATP (lanes 1, 2, 4, 7, 9, and 11) or ATPγS (lanes 5, 8, and 12). All reactions contained RPA except those in lanes 6–8, and all reactions contained primer–template DNA bound beads except that in lane 1 (beads without DNA) and those in lanes 6–9 (template DNA alone bound beads).
Mentions: Using the same relative amounts of each fraction present in the silver-stained gel (Figure 1A), the affinity-purified RSR was analyzed for RHR loading (Figure 1F). The initial fraction that was loaded on to the Rad17 affinity column was functional, as recovery of RHR with the beads was not observed in reactions that lacked DNA (Figure 1F, lane 1), the Rad17-containing fraction (Figure 1F, lane2), or nucleotide (Figure 1F, lane 3). Consistent with a requirement for Rad17 for RHR loading in vitro, the flowthrough from the affinity column that lacked Rad17 but contained other Rfc2-containing complexes (such as RFC) was inactive (Figure 1F, lanes 6–8). The purified RSR was capable of loading RHR onto DNA in a nucleotide-dependent manner, similar to the starting material (compare Figure 1A, lanes 1 and 7; Figure 1F, lanes 3–5 and 12–14). The four small RFC subunit complex, which by itself does did not bind DNA nor load RHR and PCNA onto DNA in this assay (data not shown), was also detected on DNA when Rad17 was present (Figure 1F; see also Figure 3B), further suggesting that Rfc2–5 functions in a complex with Rad17. Thus, a complex of RSR that was purified from human cells could load the RHR onto DNA in vitro.

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