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On the regulation, function, and localization of the DNA-dependent ATPase PICH.

Kaulich M, Cubizolles F, Nigg EA - Chromosoma (2012)

Bottom Line: The putative chromatin remodeling enzyme Plk1-interacting checkpoint helicase (PICH) was discovered as an interaction partner and substrate of the mitotic kinase Plk1.This work strengthens the view that PICH is an important, Plk1-regulated enzyme, whose ATPase activity is essential for maintenance of genome integrity.Although not required for the spindle assembly checkpoint, PICH is clearly important for faithful chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Growth & Development, Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4065 Basel, Switzerland.

ABSTRACT
The putative chromatin remodeling enzyme Plk1-interacting checkpoint helicase (PICH) was discovered as an interaction partner and substrate of the mitotic kinase Plk1. During mitosis PICH associates with centromeres and kinetochores and, most interestingly, constitutes a robust marker for ultrafine DNA bridges (UFBs) that connect separating chromatids in anaphase cells. The precise roles of PICH remain to be clarified. Here, we have used antibody microinjection and siRNA-rescue experiments to study PICH function and localization during M phase progression, with particular emphasis on the role of the predicted ATPase domain and the regulation of PICH localization by Plk1. We show that interference with PICH function results in chromatin bridge formation and micronucleation and that ATPase activity is critical for PICH function. Interestingly, an intact ATPase domain of PICH is required for prevention of chromatin bridge formation but not for UFB resolution, and quantitative analyses of UFB and chromatin bridge frequencies suggest that these structures are of different etiologies. We also show that the ATPase activity of PICH is required for temporal and spatial control of PICH localization to chromatin and that Plk1 likely controls PICH localization through phosphorylation of proteins distinct from PICH itself. This work strengthens the view that PICH is an important, Plk1-regulated enzyme, whose ATPase activity is essential for maintenance of genome integrity. Although not required for the spindle assembly checkpoint, PICH is clearly important for faithful chromosome segregation.

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ATPase activity is critical for PICH function and localization. a Graphic illustrating the protein domains of PICH predicted by PROSITE (Sigrist et al. 2010), with numbers indicating the start and end of each domain (amino acid position). Walker A and B motifs (key residues in brackets) as well as the major Plk1 binding site (T1063) are highlighted. bLine graph showing the ATPase activity of PICH-WT and PICH-WAB. ATPase activity was measured as a decrease in OD 340 nm over time (minutes) and displayed in arbitrary units. c Representative stills of time-lapse videos showing HeLaS3 cells stably expressing histone 2B-mCherry after co-transfection with PICH siRNA duplexes and siRNA refractory PICH rescue plasmids. T = 0 was set at the onset of anaphase and numbers indicate elapsed time (minutes). Consistent with previous data (Hubner et al. 2010), the expression of PICH-WT and PICH-WAB increased the time required from NEBD until anaphase onset, due to sequestration of Plk1. Arrows indicate chromatin bridge formation. dBar graph showing the percentage of anaphase cells showing chromatin bridges after treatment as in c. Analyses were performed on >30 cells per condition, over three independent experiments. Student’s t test revealed significance at p < 0.05. eBox-and-whisker plot showing elapsed time (minutes) from anaphase onset until dissociation from chromatin of PICH WT and WAB mutant, respectively, after treatment of cells as in c. Analyses were performed on >30 cells per condition, over three independent experiments. Lower and upper whiskers represent 10th and 90th percentiles, respectively
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Fig2: ATPase activity is critical for PICH function and localization. a Graphic illustrating the protein domains of PICH predicted by PROSITE (Sigrist et al. 2010), with numbers indicating the start and end of each domain (amino acid position). Walker A and B motifs (key residues in brackets) as well as the major Plk1 binding site (T1063) are highlighted. bLine graph showing the ATPase activity of PICH-WT and PICH-WAB. ATPase activity was measured as a decrease in OD 340 nm over time (minutes) and displayed in arbitrary units. c Representative stills of time-lapse videos showing HeLaS3 cells stably expressing histone 2B-mCherry after co-transfection with PICH siRNA duplexes and siRNA refractory PICH rescue plasmids. T = 0 was set at the onset of anaphase and numbers indicate elapsed time (minutes). Consistent with previous data (Hubner et al. 2010), the expression of PICH-WT and PICH-WAB increased the time required from NEBD until anaphase onset, due to sequestration of Plk1. Arrows indicate chromatin bridge formation. dBar graph showing the percentage of anaphase cells showing chromatin bridges after treatment as in c. Analyses were performed on >30 cells per condition, over three independent experiments. Student’s t test revealed significance at p < 0.05. eBox-and-whisker plot showing elapsed time (minutes) from anaphase onset until dissociation from chromatin of PICH WT and WAB mutant, respectively, after treatment of cells as in c. Analyses were performed on >30 cells per condition, over three independent experiments. Lower and upper whiskers represent 10th and 90th percentiles, respectively

Mentions: As predicted for a member of the SNF2/SWI class of ATPases, PICH carries Walker A and Walker B motifs that are implicated in phosphate binding and Mg2+ binding, respectively (Fig. 2a) (Baumann et al. 2007; Gorbalenya and Koonin 1993). To demonstrate the predicted ATPase activity of PICH and study its biological role, we generated a mutant (PICH-WAB) in which residues K128 (Walker A) and E229 (Walker B) were substituted by alanine (A) and glutamine (Q), respectively. Then, 293 T cells were transfected with PICH-WT or PICH-WAB and arrested in mitosis by addition of nocodazole, before the overexpressed PICH proteins were immunoprecipitated and analyzed for their ability to hydrolyze ATP in vitro. In contrast to PICH-WT, PICH-WAB did not show any in vitro ATPase activity (Fig. 2b), demonstrating that the ATPase domain of PICH is functional and requires the Walker A and B motifs. We recognize that the Walker A box substitution of lysine with alanine is likely to prevent nucleotide binding and not just hydrolysis and that many ATPases undergo a conformational change upon ATP binding (Walker et al. 1982). Thus, we emphasize that the effects reported in this study do not necessarily result from a failure of the WAB mutant to hydrolyze ATP but could also reflect an inability to undergo an ATP-dependent conformational change.Fig. 2


On the regulation, function, and localization of the DNA-dependent ATPase PICH.

Kaulich M, Cubizolles F, Nigg EA - Chromosoma (2012)

ATPase activity is critical for PICH function and localization. a Graphic illustrating the protein domains of PICH predicted by PROSITE (Sigrist et al. 2010), with numbers indicating the start and end of each domain (amino acid position). Walker A and B motifs (key residues in brackets) as well as the major Plk1 binding site (T1063) are highlighted. bLine graph showing the ATPase activity of PICH-WT and PICH-WAB. ATPase activity was measured as a decrease in OD 340 nm over time (minutes) and displayed in arbitrary units. c Representative stills of time-lapse videos showing HeLaS3 cells stably expressing histone 2B-mCherry after co-transfection with PICH siRNA duplexes and siRNA refractory PICH rescue plasmids. T = 0 was set at the onset of anaphase and numbers indicate elapsed time (minutes). Consistent with previous data (Hubner et al. 2010), the expression of PICH-WT and PICH-WAB increased the time required from NEBD until anaphase onset, due to sequestration of Plk1. Arrows indicate chromatin bridge formation. dBar graph showing the percentage of anaphase cells showing chromatin bridges after treatment as in c. Analyses were performed on >30 cells per condition, over three independent experiments. Student’s t test revealed significance at p < 0.05. eBox-and-whisker plot showing elapsed time (minutes) from anaphase onset until dissociation from chromatin of PICH WT and WAB mutant, respectively, after treatment of cells as in c. Analyses were performed on >30 cells per condition, over three independent experiments. Lower and upper whiskers represent 10th and 90th percentiles, respectively
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3401297&req=5

Fig2: ATPase activity is critical for PICH function and localization. a Graphic illustrating the protein domains of PICH predicted by PROSITE (Sigrist et al. 2010), with numbers indicating the start and end of each domain (amino acid position). Walker A and B motifs (key residues in brackets) as well as the major Plk1 binding site (T1063) are highlighted. bLine graph showing the ATPase activity of PICH-WT and PICH-WAB. ATPase activity was measured as a decrease in OD 340 nm over time (minutes) and displayed in arbitrary units. c Representative stills of time-lapse videos showing HeLaS3 cells stably expressing histone 2B-mCherry after co-transfection with PICH siRNA duplexes and siRNA refractory PICH rescue plasmids. T = 0 was set at the onset of anaphase and numbers indicate elapsed time (minutes). Consistent with previous data (Hubner et al. 2010), the expression of PICH-WT and PICH-WAB increased the time required from NEBD until anaphase onset, due to sequestration of Plk1. Arrows indicate chromatin bridge formation. dBar graph showing the percentage of anaphase cells showing chromatin bridges after treatment as in c. Analyses were performed on >30 cells per condition, over three independent experiments. Student’s t test revealed significance at p < 0.05. eBox-and-whisker plot showing elapsed time (minutes) from anaphase onset until dissociation from chromatin of PICH WT and WAB mutant, respectively, after treatment of cells as in c. Analyses were performed on >30 cells per condition, over three independent experiments. Lower and upper whiskers represent 10th and 90th percentiles, respectively
Mentions: As predicted for a member of the SNF2/SWI class of ATPases, PICH carries Walker A and Walker B motifs that are implicated in phosphate binding and Mg2+ binding, respectively (Fig. 2a) (Baumann et al. 2007; Gorbalenya and Koonin 1993). To demonstrate the predicted ATPase activity of PICH and study its biological role, we generated a mutant (PICH-WAB) in which residues K128 (Walker A) and E229 (Walker B) were substituted by alanine (A) and glutamine (Q), respectively. Then, 293 T cells were transfected with PICH-WT or PICH-WAB and arrested in mitosis by addition of nocodazole, before the overexpressed PICH proteins were immunoprecipitated and analyzed for their ability to hydrolyze ATP in vitro. In contrast to PICH-WT, PICH-WAB did not show any in vitro ATPase activity (Fig. 2b), demonstrating that the ATPase domain of PICH is functional and requires the Walker A and B motifs. We recognize that the Walker A box substitution of lysine with alanine is likely to prevent nucleotide binding and not just hydrolysis and that many ATPases undergo a conformational change upon ATP binding (Walker et al. 1982). Thus, we emphasize that the effects reported in this study do not necessarily result from a failure of the WAB mutant to hydrolyze ATP but could also reflect an inability to undergo an ATP-dependent conformational change.Fig. 2

Bottom Line: The putative chromatin remodeling enzyme Plk1-interacting checkpoint helicase (PICH) was discovered as an interaction partner and substrate of the mitotic kinase Plk1.This work strengthens the view that PICH is an important, Plk1-regulated enzyme, whose ATPase activity is essential for maintenance of genome integrity.Although not required for the spindle assembly checkpoint, PICH is clearly important for faithful chromosome segregation.

View Article: PubMed Central - PubMed

Affiliation: Growth & Development, Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4065 Basel, Switzerland.

ABSTRACT
The putative chromatin remodeling enzyme Plk1-interacting checkpoint helicase (PICH) was discovered as an interaction partner and substrate of the mitotic kinase Plk1. During mitosis PICH associates with centromeres and kinetochores and, most interestingly, constitutes a robust marker for ultrafine DNA bridges (UFBs) that connect separating chromatids in anaphase cells. The precise roles of PICH remain to be clarified. Here, we have used antibody microinjection and siRNA-rescue experiments to study PICH function and localization during M phase progression, with particular emphasis on the role of the predicted ATPase domain and the regulation of PICH localization by Plk1. We show that interference with PICH function results in chromatin bridge formation and micronucleation and that ATPase activity is critical for PICH function. Interestingly, an intact ATPase domain of PICH is required for prevention of chromatin bridge formation but not for UFB resolution, and quantitative analyses of UFB and chromatin bridge frequencies suggest that these structures are of different etiologies. We also show that the ATPase activity of PICH is required for temporal and spatial control of PICH localization to chromatin and that Plk1 likely controls PICH localization through phosphorylation of proteins distinct from PICH itself. This work strengthens the view that PICH is an important, Plk1-regulated enzyme, whose ATPase activity is essential for maintenance of genome integrity. Although not required for the spindle assembly checkpoint, PICH is clearly important for faithful chromosome segregation.

Show MeSH
Related in: MedlinePlus