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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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PCNA and RHR Loading Are Specifically Stimulated by Human RPAIn both (A) and (B), lanes represent loading reactions performed as described in the Materials and Methods with either 5′ recessed or 3′ recessed primer–template DNA–RPA complex bound to beads, and recovery of proteins with the beads was analyzed by Wb for the indicated proteins. In (A), PCNA loading was assayed in the absence of RPA (lane 2) or in the presence of the indicated amounts of either yeast RPA (lanes 3–5) or human RPA (lanes 6–9). All reactions contained 3′ recessed primer–template DNA (except for that in lane 1), 2 pmol of PCNA, 0.25 pmol of RFC, and ATP. In (B), RHR loading was assayed in the absence of RPA (lane 2) or in the presence of the indicated amounts of either yeast RPA (lanes 3 and 4) or human RPA (lanes 5 and 6). All reactions contained 5′ recessed primer–template DNA (except for that in lane 1), 1 pmol of RHR complex, 0.25 pmol of RSR, and ATP.
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pbio.0000033-g006: PCNA and RHR Loading Are Specifically Stimulated by Human RPAIn both (A) and (B), lanes represent loading reactions performed as described in the Materials and Methods with either 5′ recessed or 3′ recessed primer–template DNA–RPA complex bound to beads, and recovery of proteins with the beads was analyzed by Wb for the indicated proteins. In (A), PCNA loading was assayed in the absence of RPA (lane 2) or in the presence of the indicated amounts of either yeast RPA (lanes 3–5) or human RPA (lanes 6–9). All reactions contained 3′ recessed primer–template DNA (except for that in lane 1), 2 pmol of PCNA, 0.25 pmol of RFC, and ATP. In (B), RHR loading was assayed in the absence of RPA (lane 2) or in the presence of the indicated amounts of either yeast RPA (lanes 3 and 4) or human RPA (lanes 5 and 6). All reactions contained 5′ recessed primer–template DNA (except for that in lane 1), 1 pmol of RHR complex, 0.25 pmol of RSR, and ATP.

Mentions: When PCNA loading was analyzed in the presence of saturating levels of either yeast (Figure 6A, lanes 3–5) or human RPA (Figure 6A, lanes 6–9), recovery of the PCNA was poorer in the presence of yeast RPA compared to human RPA. Inefficient recovery of the PCNA was not due to an inability of yeast RPA to prevent human PCNA clamp sliding, because usage of alternative recessed 3′ end substrates in this assay permitted efficient and comparable PCNA recovery in the presence of yeast and human RPA (V. E. and B. S., unpublished data). Hence, recovery of the PCNA in the presence of yeast RPA was DNA structure specific and not due to an intrinsic (1) inability of yeast RPA to prevent human PCNA clamp sliding nor (2) human RFC inhibitory activity of yeast RPA. Therefore, we concluded that RPA performed two functions in our assay: first, it prevented the clamp from sliding off the DNA, and second, it specifically stimulated the activity of RFC on canonical primer–template recessed 3′ end DNA substrates. Likewise, when yeast RPA was tested for the ability to support RHR loading (Figure 6B), RSR activity was found to be preferentially stimulated by human RPA (Figure 6B, lanes 5 and 6) compared to yeast RPA (Figure 6B, lanes 3 and 4). Thus, the clamp loader functions of both RFC and RSR were modulated by interactions with RPA.


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)

PCNA and RHR Loading Are Specifically Stimulated by Human RPAIn both (A) and (B), lanes represent loading reactions performed as described in the Materials and Methods with either 5′ recessed or 3′ recessed primer–template DNA–RPA complex bound to beads, and recovery of proteins with the beads was analyzed by Wb for the indicated proteins. In (A), PCNA loading was assayed in the absence of RPA (lane 2) or in the presence of the indicated amounts of either yeast RPA (lanes 3–5) or human RPA (lanes 6–9). All reactions contained 3′ recessed primer–template DNA (except for that in lane 1), 2 pmol of PCNA, 0.25 pmol of RFC, and ATP. In (B), RHR loading was assayed in the absence of RPA (lane 2) or in the presence of the indicated amounts of either yeast RPA (lanes 3 and 4) or human RPA (lanes 5 and 6). All reactions contained 5′ recessed primer–template DNA (except for that in lane 1), 1 pmol of RHR complex, 0.25 pmol of RSR, and ATP.
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pbio.0000033-g006: PCNA and RHR Loading Are Specifically Stimulated by Human RPAIn both (A) and (B), lanes represent loading reactions performed as described in the Materials and Methods with either 5′ recessed or 3′ recessed primer–template DNA–RPA complex bound to beads, and recovery of proteins with the beads was analyzed by Wb for the indicated proteins. In (A), PCNA loading was assayed in the absence of RPA (lane 2) or in the presence of the indicated amounts of either yeast RPA (lanes 3–5) or human RPA (lanes 6–9). All reactions contained 3′ recessed primer–template DNA (except for that in lane 1), 2 pmol of PCNA, 0.25 pmol of RFC, and ATP. In (B), RHR loading was assayed in the absence of RPA (lane 2) or in the presence of the indicated amounts of either yeast RPA (lanes 3 and 4) or human RPA (lanes 5 and 6). All reactions contained 5′ recessed primer–template DNA (except for that in lane 1), 1 pmol of RHR complex, 0.25 pmol of RSR, and ATP.
Mentions: When PCNA loading was analyzed in the presence of saturating levels of either yeast (Figure 6A, lanes 3–5) or human RPA (Figure 6A, lanes 6–9), recovery of the PCNA was poorer in the presence of yeast RPA compared to human RPA. Inefficient recovery of the PCNA was not due to an inability of yeast RPA to prevent human PCNA clamp sliding, because usage of alternative recessed 3′ end substrates in this assay permitted efficient and comparable PCNA recovery in the presence of yeast and human RPA (V. E. and B. S., unpublished data). Hence, recovery of the PCNA in the presence of yeast RPA was DNA structure specific and not due to an intrinsic (1) inability of yeast RPA to prevent human PCNA clamp sliding nor (2) human RFC inhibitory activity of yeast RPA. Therefore, we concluded that RPA performed two functions in our assay: first, it prevented the clamp from sliding off the DNA, and second, it specifically stimulated the activity of RFC on canonical primer–template recessed 3′ end DNA substrates. Likewise, when yeast RPA was tested for the ability to support RHR loading (Figure 6B), RSR activity was found to be preferentially stimulated by human RPA (Figure 6B, lanes 5 and 6) compared to yeast RPA (Figure 6B, lanes 3 and 4). Thus, the clamp loader functions of both RFC and RSR were modulated by interactions with RPA.

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