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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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Purified RSR Is an ATPase That Is Poorly Stimulated by DNA(A) Titration of purified RSR and RFC. Visualized by SDS-PAGE and silver staining were 0.3, 0.15, and 0.075 pmol of RSR (lanes 1–3) and RFC (lanes 4–6).(B) ATPase activity of the indicated amount of either RSR (squares) or RFC (circles) was measured after 60 min in the presence of 200 nM 5′ and 3′ recessed primer–template DNA.(C) ATPase activity of either 0.3 pmol of RSR (squares) or 0.30 pmol of RFC (circles) was analyzed after a 60 min incubation in the absence or presence of 1.6 nM, 8 nM, 40 nM, or 200 nM 5′ and 3′ recessed primer–template DNA.(D) ATPase activity of 0.15 pmol of RSR (diamonds and circles) or 0.15 pmol of RFC (triangles and squares) in the absence of DNA (diamonds and triangles) or presence of 200 nM 5′ and 3′ recessed primer–template DNA was measured after either a 3.5, 7.5, 15, 30, or 60 min incubation. All reactions were performed as described in the Materials and Methods.
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pbio.0000033-g002: Purified RSR Is an ATPase That Is Poorly Stimulated by DNA(A) Titration of purified RSR and RFC. Visualized by SDS-PAGE and silver staining were 0.3, 0.15, and 0.075 pmol of RSR (lanes 1–3) and RFC (lanes 4–6).(B) ATPase activity of the indicated amount of either RSR (squares) or RFC (circles) was measured after 60 min in the presence of 200 nM 5′ and 3′ recessed primer–template DNA.(C) ATPase activity of either 0.3 pmol of RSR (squares) or 0.30 pmol of RFC (circles) was analyzed after a 60 min incubation in the absence or presence of 1.6 nM, 8 nM, 40 nM, or 200 nM 5′ and 3′ recessed primer–template DNA.(D) ATPase activity of 0.15 pmol of RSR (diamonds and circles) or 0.15 pmol of RFC (triangles and squares) in the absence of DNA (diamonds and triangles) or presence of 200 nM 5′ and 3′ recessed primer–template DNA was measured after either a 3.5, 7.5, 15, 30, or 60 min incubation. All reactions were performed as described in the Materials and Methods.

Mentions: RFC is a DNA-activated ATPase that is preferentially activated by primer–template DNAs (Tsurimoto and Stillman 1990). When RSR was examined along with an equivalent amount of purified recombinant RFC for ATPase activity in the presence of the primer–template substrate (Figure 2), or poly(dA):oligo(dT) (data not shown), the ATPase activity of the complex was observed reproducibly to be stimulated no more than 2-fold by DNA. This was in sharp contrast to similar amounts of the RFC ATPase that were stimulated by DNA to greater than 10-fold (Cai et al. 1997; Podust and Fanning 1997; Ellison and Stillman 1998). The observation, also reported for the yeast checkpoint clamp loader, was surprising, particularly since the ATPase activities of both the Rfc2–5 and the Rfc2–4 subcomplexes are significantly stimulated by DNA (Podust et al. 1998; V. E. and B. S., unpublished data). These data suggest that within the RSR complex the Rad17 subunit altered the ATPase cycle of the Rfc2–5 subunits.


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)

Purified RSR Is an ATPase That Is Poorly Stimulated by DNA(A) Titration of purified RSR and RFC. Visualized by SDS-PAGE and silver staining were 0.3, 0.15, and 0.075 pmol of RSR (lanes 1–3) and RFC (lanes 4–6).(B) ATPase activity of the indicated amount of either RSR (squares) or RFC (circles) was measured after 60 min in the presence of 200 nM 5′ and 3′ recessed primer–template DNA.(C) ATPase activity of either 0.3 pmol of RSR (squares) or 0.30 pmol of RFC (circles) was analyzed after a 60 min incubation in the absence or presence of 1.6 nM, 8 nM, 40 nM, or 200 nM 5′ and 3′ recessed primer–template DNA.(D) ATPase activity of 0.15 pmol of RSR (diamonds and circles) or 0.15 pmol of RFC (triangles and squares) in the absence of DNA (diamonds and triangles) or presence of 200 nM 5′ and 3′ recessed primer–template DNA was measured after either a 3.5, 7.5, 15, 30, or 60 min incubation. All reactions were performed as described in the Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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

pbio.0000033-g002: Purified RSR Is an ATPase That Is Poorly Stimulated by DNA(A) Titration of purified RSR and RFC. Visualized by SDS-PAGE and silver staining were 0.3, 0.15, and 0.075 pmol of RSR (lanes 1–3) and RFC (lanes 4–6).(B) ATPase activity of the indicated amount of either RSR (squares) or RFC (circles) was measured after 60 min in the presence of 200 nM 5′ and 3′ recessed primer–template DNA.(C) ATPase activity of either 0.3 pmol of RSR (squares) or 0.30 pmol of RFC (circles) was analyzed after a 60 min incubation in the absence or presence of 1.6 nM, 8 nM, 40 nM, or 200 nM 5′ and 3′ recessed primer–template DNA.(D) ATPase activity of 0.15 pmol of RSR (diamonds and circles) or 0.15 pmol of RFC (triangles and squares) in the absence of DNA (diamonds and triangles) or presence of 200 nM 5′ and 3′ recessed primer–template DNA was measured after either a 3.5, 7.5, 15, 30, or 60 min incubation. All reactions were performed as described in the Materials and Methods.
Mentions: RFC is a DNA-activated ATPase that is preferentially activated by primer–template DNAs (Tsurimoto and Stillman 1990). When RSR was examined along with an equivalent amount of purified recombinant RFC for ATPase activity in the presence of the primer–template substrate (Figure 2), or poly(dA):oligo(dT) (data not shown), the ATPase activity of the complex was observed reproducibly to be stimulated no more than 2-fold by DNA. This was in sharp contrast to similar amounts of the RFC ATPase that were stimulated by DNA to greater than 10-fold (Cai et al. 1997; Podust and Fanning 1997; Ellison and Stillman 1998). The observation, also reported for the yeast checkpoint clamp loader, was surprising, particularly since the ATPase activities of both the Rfc2–5 and the Rfc2–4 subcomplexes are significantly stimulated by DNA (Podust et al. 1998; V. E. and B. S., unpublished data). These data suggest that within the RSR complex the Rad17 subunit altered the ATPase cycle of the Rfc2–5 subunits.

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