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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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The Purified Human RSR Complex Can Load the RHR Complex onto DNA In Vitro(A) RSR was purified from the Rfc2 Ab affinity column eluate by anti-Rad17 Ab affinity chromatography, and the peptide-eluted material was concentrated by Q–sepharose chromatography. An equivalent volume (5 μl) of the load onto the anti-Rad17 column (lane 1, labeled L), the flowthrough from the column (lane 2, labeled FT), each peptide elution fraction (lanes 3–5), and the indicated amounts of the concentrated, purified complex (lanes 6–8) were analyzed by silver staining and Wb for Rad17.(B) The same fractions present in the silver-stained gel in (A) were analyzed by Wb for Rfc1, Ctf18, Rfc2, Rfc4, and Rfc5, and the lanes are as loaded and numbered in (A).(C) Peptide sequences for the proteins present in the purified Q–sepharose fraction.(D) Purification of the RHR. RHR was purified from E. coli by Talon affinity, Q–sepharose, and phosphocellulose chromatography and by glycerol gradient sedimentation (shown here). The load onto the gradient (lane L) and fractions (corresponding to lane numbers) as well as any material in the pellet (lane B) were analyzed by silver staining (shown) and Wb (not shown). Arrows indicate the sedimentation position of protein standards from a gradient prepared in parallel.(E) Assay for RHR and PCNA loading. RHR and PCNA loading were examined by monitoring the binding of the proteins to a DNA–RPA complex bound to streptavidin–agarose beads by Wb of the bead-bound fractions. The DNA substrate consist of a 90 nucleotide (nt) 3′ biotinylated template and 30 nt primer positioned in the center of the template, resulting in a substrate with 30 nt single-stranded recessed 5′ and 3′ ends to which RPA was bound.(F) RSR is sufficient to load RHR onto DNA in vitro. Reactions were performed as described in the Materials and Methods, and the bead-precipitated products were analyzed by Wb for Rad17, Rfc2, Rad9, Hus1, and Rad1. The fractions from the purification shown in (A) were assayed for RHR-loading activity. Lanes represent reactions that contained the same amount of the anti-Rad17 Ab column load (lanes 1, 3, 4, and 5), flowthrough (lanes 6–8), and the Q–sepharose concentrated protein (lanes 9–17) as shown in the silver-stained gel in (A), or no source of Rad17 (buffer only, lane 2). All reactions contained 5′ and 3′ recessed primer–template DNA–RPA complex bound to beads (except for that in lane 1, which contained beads alone), 1 pmol of RHR complex, and the indicated nucleotide cofactor (ATP: lanes 1, 2, 4, 7, 10, 13, and 16; ATPγS: lanes 5, 8, 11, 14, and 17) or no nucleotide (lanes 3, 6, 9, 12, and 15). The lane labeled L represents 20% of the input of RHR and anti-Rad17 column load used in the reaction.
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pbio.0000033-g001: The Purified Human RSR Complex Can Load the RHR Complex onto DNA In Vitro(A) RSR was purified from the Rfc2 Ab affinity column eluate by anti-Rad17 Ab affinity chromatography, and the peptide-eluted material was concentrated by Q–sepharose chromatography. An equivalent volume (5 μl) of the load onto the anti-Rad17 column (lane 1, labeled L), the flowthrough from the column (lane 2, labeled FT), each peptide elution fraction (lanes 3–5), and the indicated amounts of the concentrated, purified complex (lanes 6–8) were analyzed by silver staining and Wb for Rad17.(B) The same fractions present in the silver-stained gel in (A) were analyzed by Wb for Rfc1, Ctf18, Rfc2, Rfc4, and Rfc5, and the lanes are as loaded and numbered in (A).(C) Peptide sequences for the proteins present in the purified Q–sepharose fraction.(D) Purification of the RHR. RHR was purified from E. coli by Talon affinity, Q–sepharose, and phosphocellulose chromatography and by glycerol gradient sedimentation (shown here). The load onto the gradient (lane L) and fractions (corresponding to lane numbers) as well as any material in the pellet (lane B) were analyzed by silver staining (shown) and Wb (not shown). Arrows indicate the sedimentation position of protein standards from a gradient prepared in parallel.(E) Assay for RHR and PCNA loading. RHR and PCNA loading were examined by monitoring the binding of the proteins to a DNA–RPA complex bound to streptavidin–agarose beads by Wb of the bead-bound fractions. The DNA substrate consist of a 90 nucleotide (nt) 3′ biotinylated template and 30 nt primer positioned in the center of the template, resulting in a substrate with 30 nt single-stranded recessed 5′ and 3′ ends to which RPA was bound.(F) RSR is sufficient to load RHR onto DNA in vitro. Reactions were performed as described in the Materials and Methods, and the bead-precipitated products were analyzed by Wb for Rad17, Rfc2, Rad9, Hus1, and Rad1. The fractions from the purification shown in (A) were assayed for RHR-loading activity. Lanes represent reactions that contained the same amount of the anti-Rad17 Ab column load (lanes 1, 3, 4, and 5), flowthrough (lanes 6–8), and the Q–sepharose concentrated protein (lanes 9–17) as shown in the silver-stained gel in (A), or no source of Rad17 (buffer only, lane 2). All reactions contained 5′ and 3′ recessed primer–template DNA–RPA complex bound to beads (except for that in lane 1, which contained beads alone), 1 pmol of RHR complex, and the indicated nucleotide cofactor (ATP: lanes 1, 2, 4, 7, 10, 13, and 16; ATPγS: lanes 5, 8, 11, 14, and 17) or no nucleotide (lanes 3, 6, 9, 12, and 15). The lane labeled L represents 20% of the input of RHR and anti-Rad17 column load used in the reaction.

Mentions: Using the Ab described above, Rad17 was purified from the partially purified fraction by anti-Rad17 affinity followed by Q–sepharose chromatography (Figure 1A). In addition to Rad17 itself, copurification of Rfc2–5 was confirmed by Western blotting (Wb) and mass spectrometry analyses (Figure 1B and 1C, respectively). Thus, using sequential Rfc2 and Rad17 Ab affinity chromatography, we identified a highly purified RSR complex (Rad17 and Rfc2–5; see Table 1 for yeast orthologs). Other proteins in the starting fraction, including Rfc1 and Ctf18, appeared to be components of unique Rfc2-containing complexes, for these proteins were recovered in the anti-Rad17 column flowthrough and therefore did not copurify with Rad17.


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)

The Purified Human RSR Complex Can Load the RHR Complex onto DNA In Vitro(A) RSR was purified from the Rfc2 Ab affinity column eluate by anti-Rad17 Ab affinity chromatography, and the peptide-eluted material was concentrated by Q–sepharose chromatography. An equivalent volume (5 μl) of the load onto the anti-Rad17 column (lane 1, labeled L), the flowthrough from the column (lane 2, labeled FT), each peptide elution fraction (lanes 3–5), and the indicated amounts of the concentrated, purified complex (lanes 6–8) were analyzed by silver staining and Wb for Rad17.(B) The same fractions present in the silver-stained gel in (A) were analyzed by Wb for Rfc1, Ctf18, Rfc2, Rfc4, and Rfc5, and the lanes are as loaded and numbered in (A).(C) Peptide sequences for the proteins present in the purified Q–sepharose fraction.(D) Purification of the RHR. RHR was purified from E. coli by Talon affinity, Q–sepharose, and phosphocellulose chromatography and by glycerol gradient sedimentation (shown here). The load onto the gradient (lane L) and fractions (corresponding to lane numbers) as well as any material in the pellet (lane B) were analyzed by silver staining (shown) and Wb (not shown). Arrows indicate the sedimentation position of protein standards from a gradient prepared in parallel.(E) Assay for RHR and PCNA loading. RHR and PCNA loading were examined by monitoring the binding of the proteins to a DNA–RPA complex bound to streptavidin–agarose beads by Wb of the bead-bound fractions. The DNA substrate consist of a 90 nucleotide (nt) 3′ biotinylated template and 30 nt primer positioned in the center of the template, resulting in a substrate with 30 nt single-stranded recessed 5′ and 3′ ends to which RPA was bound.(F) RSR is sufficient to load RHR onto DNA in vitro. Reactions were performed as described in the Materials and Methods, and the bead-precipitated products were analyzed by Wb for Rad17, Rfc2, Rad9, Hus1, and Rad1. The fractions from the purification shown in (A) were assayed for RHR-loading activity. Lanes represent reactions that contained the same amount of the anti-Rad17 Ab column load (lanes 1, 3, 4, and 5), flowthrough (lanes 6–8), and the Q–sepharose concentrated protein (lanes 9–17) as shown in the silver-stained gel in (A), or no source of Rad17 (buffer only, lane 2). All reactions contained 5′ and 3′ recessed primer–template DNA–RPA complex bound to beads (except for that in lane 1, which contained beads alone), 1 pmol of RHR complex, and the indicated nucleotide cofactor (ATP: lanes 1, 2, 4, 7, 10, 13, and 16; ATPγS: lanes 5, 8, 11, 14, and 17) or no nucleotide (lanes 3, 6, 9, 12, and 15). The lane labeled L represents 20% of the input of RHR and anti-Rad17 column load used in the reaction.
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pbio.0000033-g001: The Purified Human RSR Complex Can Load the RHR Complex onto DNA In Vitro(A) RSR was purified from the Rfc2 Ab affinity column eluate by anti-Rad17 Ab affinity chromatography, and the peptide-eluted material was concentrated by Q–sepharose chromatography. An equivalent volume (5 μl) of the load onto the anti-Rad17 column (lane 1, labeled L), the flowthrough from the column (lane 2, labeled FT), each peptide elution fraction (lanes 3–5), and the indicated amounts of the concentrated, purified complex (lanes 6–8) were analyzed by silver staining and Wb for Rad17.(B) The same fractions present in the silver-stained gel in (A) were analyzed by Wb for Rfc1, Ctf18, Rfc2, Rfc4, and Rfc5, and the lanes are as loaded and numbered in (A).(C) Peptide sequences for the proteins present in the purified Q–sepharose fraction.(D) Purification of the RHR. RHR was purified from E. coli by Talon affinity, Q–sepharose, and phosphocellulose chromatography and by glycerol gradient sedimentation (shown here). The load onto the gradient (lane L) and fractions (corresponding to lane numbers) as well as any material in the pellet (lane B) were analyzed by silver staining (shown) and Wb (not shown). Arrows indicate the sedimentation position of protein standards from a gradient prepared in parallel.(E) Assay for RHR and PCNA loading. RHR and PCNA loading were examined by monitoring the binding of the proteins to a DNA–RPA complex bound to streptavidin–agarose beads by Wb of the bead-bound fractions. The DNA substrate consist of a 90 nucleotide (nt) 3′ biotinylated template and 30 nt primer positioned in the center of the template, resulting in a substrate with 30 nt single-stranded recessed 5′ and 3′ ends to which RPA was bound.(F) RSR is sufficient to load RHR onto DNA in vitro. Reactions were performed as described in the Materials and Methods, and the bead-precipitated products were analyzed by Wb for Rad17, Rfc2, Rad9, Hus1, and Rad1. The fractions from the purification shown in (A) were assayed for RHR-loading activity. Lanes represent reactions that contained the same amount of the anti-Rad17 Ab column load (lanes 1, 3, 4, and 5), flowthrough (lanes 6–8), and the Q–sepharose concentrated protein (lanes 9–17) as shown in the silver-stained gel in (A), or no source of Rad17 (buffer only, lane 2). All reactions contained 5′ and 3′ recessed primer–template DNA–RPA complex bound to beads (except for that in lane 1, which contained beads alone), 1 pmol of RHR complex, and the indicated nucleotide cofactor (ATP: lanes 1, 2, 4, 7, 10, 13, and 16; ATPγS: lanes 5, 8, 11, 14, and 17) or no nucleotide (lanes 3, 6, 9, 12, and 15). The lane labeled L represents 20% of the input of RHR and anti-Rad17 column load used in the reaction.
Mentions: Using the Ab described above, Rad17 was purified from the partially purified fraction by anti-Rad17 affinity followed by Q–sepharose chromatography (Figure 1A). In addition to Rad17 itself, copurification of Rfc2–5 was confirmed by Western blotting (Wb) and mass spectrometry analyses (Figure 1B and 1C, respectively). Thus, using sequential Rfc2 and Rad17 Ab affinity chromatography, we identified a highly purified RSR complex (Rad17 and Rfc2–5; see Table 1 for yeast orthologs). Other proteins in the starting fraction, including Rfc1 and Ctf18, appeared to be components of unique Rfc2-containing complexes, for these proteins were recovered in the anti-Rad17 column flowthrough and therefore did not copurify with Rad17.

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