Limits...
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.

Show MeSH

Related in: MedlinePlus

Opposite DNA Substrate Preference Exhibited by RFC and RSRLoading reactions in (A) and (B) were performed as described in the Materials and Methods with DNA substrates bound to beads, and recovery of proteins with the beads was analyzed by Wb for the indicated proteins. Lanes represent reactions using either a 5′ and 3′ recessed primer–template DNA (lanes 2–5), a 3′ recessed primer–template DNA (lanes 6–9), a 5′ recessed primer–template DNA (lanes 10–13), or no DNA (lane 1). All reactions contained RPA and the indicated nucleotide (ATP: lanes 1, 2, 4, 6, 8, 10, and 12; ATPγS: lanes 5, 9, and 13) or no nucleotide (lanes 3, 7, and 11). In (A), all reactions contained 0.25 pmol of RFC (except for those in lanes 2, 6, and 10) and 2 pmol of PCNA. In (B), all reactions contained 0.25 pmol of RSR (except for those in lanes 2, 6, and 10) and 1 pmol of RHR. In both (A) and (B), 20% of the input in each reaction is represented in the last lane in each panel.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC261875&req=5

pbio.0000033-g004: Opposite DNA Substrate Preference Exhibited by RFC and RSRLoading reactions in (A) and (B) were performed as described in the Materials and Methods with DNA substrates bound to beads, and recovery of proteins with the beads was analyzed by Wb for the indicated proteins. Lanes represent reactions using either a 5′ and 3′ recessed primer–template DNA (lanes 2–5), a 3′ recessed primer–template DNA (lanes 6–9), a 5′ recessed primer–template DNA (lanes 10–13), or no DNA (lane 1). All reactions contained RPA and the indicated nucleotide (ATP: lanes 1, 2, 4, 6, 8, 10, and 12; ATPγS: lanes 5, 9, and 13) or no nucleotide (lanes 3, 7, and 11). In (A), all reactions contained 0.25 pmol of RFC (except for those in lanes 2, 6, and 10) and 2 pmol of PCNA. In (B), all reactions contained 0.25 pmol of RSR (except for those in lanes 2, 6, and 10) and 1 pmol of RHR. In both (A) and (B), 20% of the input in each reaction is represented in the last lane in each panel.

Mentions: The primer–template substrate used had both a recessed 5′ end and a recessed 3′ end because the primer was located in the center of the template. Although it is well established that RFC loads PCNA onto DNA containing either a recessed 3′ end or a nicked double-strand DNA, usage of alternative DNA structures as substrates, such as those with recessed 5′ ends, has not been examined (Mossi and Hubscher 1998). Hence, we tested the ability of RFC and RSR to load their respective clamps onto DNA containing either a recessed 5′ end or a recessed 3′ end. As expected, we observed a requirement for a DNA substrate with a recessed 3′ end for PCNA loading by RFC; however, loading onto a recessed 5′ end DNA substrate was essentially undetectable (Figure 4A, lanes 12 and 13). Unexpectedly, however, RSR possessed the opposite substrate specificity (Figure 4B), since a DNA substrate with a recessed 5′ end was discovered to be a much better substrate for RHR loading (Figure 4B, lanes 11 and 12) compared to the recessed 3′ end substrate (Figure 4B, lanes 8 and 9). Thus, RSR and RFC loaded their respective clamps onto different DNA structures, relegating these clamp loaders to distinct DNA replication and repair reactions in the cell.


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)

Opposite DNA Substrate Preference Exhibited by RFC and RSRLoading reactions in (A) and (B) were performed as described in the Materials and Methods with DNA substrates bound to beads, and recovery of proteins with the beads was analyzed by Wb for the indicated proteins. Lanes represent reactions using either a 5′ and 3′ recessed primer–template DNA (lanes 2–5), a 3′ recessed primer–template DNA (lanes 6–9), a 5′ recessed primer–template DNA (lanes 10–13), or no DNA (lane 1). All reactions contained RPA and the indicated nucleotide (ATP: lanes 1, 2, 4, 6, 8, 10, and 12; ATPγS: lanes 5, 9, and 13) or no nucleotide (lanes 3, 7, and 11). In (A), all reactions contained 0.25 pmol of RFC (except for those in lanes 2, 6, and 10) and 2 pmol of PCNA. In (B), all reactions contained 0.25 pmol of RSR (except for those in lanes 2, 6, and 10) and 1 pmol of RHR. In both (A) and (B), 20% of the input in each reaction is represented in the last lane in each panel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC261875&req=5

pbio.0000033-g004: Opposite DNA Substrate Preference Exhibited by RFC and RSRLoading reactions in (A) and (B) were performed as described in the Materials and Methods with DNA substrates bound to beads, and recovery of proteins with the beads was analyzed by Wb for the indicated proteins. Lanes represent reactions using either a 5′ and 3′ recessed primer–template DNA (lanes 2–5), a 3′ recessed primer–template DNA (lanes 6–9), a 5′ recessed primer–template DNA (lanes 10–13), or no DNA (lane 1). All reactions contained RPA and the indicated nucleotide (ATP: lanes 1, 2, 4, 6, 8, 10, and 12; ATPγS: lanes 5, 9, and 13) or no nucleotide (lanes 3, 7, and 11). In (A), all reactions contained 0.25 pmol of RFC (except for those in lanes 2, 6, and 10) and 2 pmol of PCNA. In (B), all reactions contained 0.25 pmol of RSR (except for those in lanes 2, 6, and 10) and 1 pmol of RHR. In both (A) and (B), 20% of the input in each reaction is represented in the last lane in each panel.
Mentions: The primer–template substrate used had both a recessed 5′ end and a recessed 3′ end because the primer was located in the center of the template. Although it is well established that RFC loads PCNA onto DNA containing either a recessed 3′ end or a nicked double-strand DNA, usage of alternative DNA structures as substrates, such as those with recessed 5′ ends, has not been examined (Mossi and Hubscher 1998). Hence, we tested the ability of RFC and RSR to load their respective clamps onto DNA containing either a recessed 5′ end or a recessed 3′ end. As expected, we observed a requirement for a DNA substrate with a recessed 3′ end for PCNA loading by RFC; however, loading onto a recessed 5′ end DNA substrate was essentially undetectable (Figure 4A, lanes 12 and 13). Unexpectedly, however, RSR possessed the opposite substrate specificity (Figure 4B), since a DNA substrate with a recessed 5′ end was discovered to be a much better substrate for RHR loading (Figure 4B, lanes 11 and 12) compared to the recessed 3′ end substrate (Figure 4B, lanes 8 and 9). Thus, RSR and RFC loaded their respective clamps onto different DNA structures, relegating these clamp loaders to distinct DNA replication and repair reactions in the cell.

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