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Complex activities of the human Bloom's syndrome helicase are encoded in a core region comprising the RecA and Zn-binding domains.

Gyimesi M, Harami GM, Sarlós K, Hazai E, Bikádi Z, Kovács M - Nucleic Acids Res. (2012)

Bottom Line: We performed a quantitative mechanistic analysis of truncated BLM constructs that are shorter than the previously identified minimal functional module.Surprisingly, we found that a BLM construct comprising only the two conserved RecA domains and the Zn(2+)-binding domain (residues 642-1077) can efficiently perform all mentioned HR-related activities.The results demonstrate that the Zn(2+)-binding domain is necessary for functional interaction with DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, ELTE-MTA Momentum Motor Enzymology Research Group, Eötvös University, Pázmány P. s. 1/c, H-1117 Budapest, Hungary.

ABSTRACT
Bloom's syndrome DNA helicase (BLM), a member of the RecQ family, is a key player in homologous recombination (HR)-based error-free DNA repair processes. During HR, BLM exerts various biochemical activities including single-stranded (ss) DNA translocation, separation and annealing of complementary DNA strands, disruption of complex DNA structures (e.g. displacement loops) and contributes to quality control of HR via clearance of Rad51 nucleoprotein filaments. We performed a quantitative mechanistic analysis of truncated BLM constructs that are shorter than the previously identified minimal functional module. Surprisingly, we found that a BLM construct comprising only the two conserved RecA domains and the Zn(2+)-binding domain (residues 642-1077) can efficiently perform all mentioned HR-related activities. The results demonstrate that the Zn(2+)-binding domain is necessary for functional interaction with DNA. We show that the extensions of this core, including the winged-helix domain and the strand separation hairpin identified therein in other RecQ-family helicases, are not required for mechanochemical activity per se and may instead play modulatory roles and mediate protein-protein interactions.

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Nucleoprotein filament clearance by BLM constructs. (A) Steady-state ATPase activities of 20 nM BLM1077 (filled squqre), BLM1290 (open circle) and BLMFL (open triangle) measured at 25°C in the presence of 100 nM dT54 and at different hRad51 concentrations. Hill-equation () was used for data fitting in which k0 and kINH are BLM ATPase activities in the absence of hRad51 and in the presence of saturating hRad51 concentration, respectively; cINH is hRad51 concentration; K is the Michaelis constant and n is the cooperativity factor. Obtained values of K and n (not shown) were similar for all three constructs. (B) Steady-state BLM ATPase activities measured as in (A) except using SSB instead of hRad51. SSB tetramer concentrations are indicated. (C) Steady-state ATPase activities of BLM constructs in the absence of DNA (kbasal, black) and in the presence of saturating concentrations of hRad51 and SSB (kINH,hRad51, grey and kINH,SSB, light grey, respectively, cf. panels A and B). Note that kINH,hRad51 was markedly elevated compared to kbasal in all three BLM constructs, indicating hRad51 filament clearance activity. Conversely, kINH,SSB was practically identical to kbasal in BLMFL and BLM1290, but markedly elevated in BLM1077, indicating that only the latter construct is able to clear SSB from ssDNA. (D) Kinetics of hRad51 nucleoprotein filament clearance by BLM constructs, as monitored by MDCC-SSB fluorescence. dT54 (150 nM) was incubated with 2.8 µM hRad51 for 30 min on ice. BLM constructs were then added at a concentration of 200 nM, and samples were incubated for additional 10 min on ice. Traces labelled BLM1077 (black), BLM1290 (grey) and BLMFL (light grey) were recorded upon rapidly mixing dT54.hRad51.BLM premixtures with 1 µM MDDC-SSB (marked as SSB*) plus 2 mM ATP to monitor hRad51 clearance by BLM. The panel also shows control traces recorded upon mixing MDCC-SSB with buffer alone, MDCC-SSB with free dT54 (in the absence of hRad51 and BLM), and dT54.Rad51.BLM premixtures with MDCC-SSB in the absence of ATP. (In the latter case, traces were very similar in the case of all BLM constructs and also when BLM was omitted.) Traces were corrected for MDCC-SSB fluorescence levels at the start of the reactions. The extent of active hRad51 clearance by BLM, as calculated from the final steady-state MDCC-SSB fluorescence levels, was 41, 41 and 39% in the case of BLM1077, BLM1290 and BLMFL, respectively.
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gks008-F6: Nucleoprotein filament clearance by BLM constructs. (A) Steady-state ATPase activities of 20 nM BLM1077 (filled squqre), BLM1290 (open circle) and BLMFL (open triangle) measured at 25°C in the presence of 100 nM dT54 and at different hRad51 concentrations. Hill-equation () was used for data fitting in which k0 and kINH are BLM ATPase activities in the absence of hRad51 and in the presence of saturating hRad51 concentration, respectively; cINH is hRad51 concentration; K is the Michaelis constant and n is the cooperativity factor. Obtained values of K and n (not shown) were similar for all three constructs. (B) Steady-state BLM ATPase activities measured as in (A) except using SSB instead of hRad51. SSB tetramer concentrations are indicated. (C) Steady-state ATPase activities of BLM constructs in the absence of DNA (kbasal, black) and in the presence of saturating concentrations of hRad51 and SSB (kINH,hRad51, grey and kINH,SSB, light grey, respectively, cf. panels A and B). Note that kINH,hRad51 was markedly elevated compared to kbasal in all three BLM constructs, indicating hRad51 filament clearance activity. Conversely, kINH,SSB was practically identical to kbasal in BLMFL and BLM1290, but markedly elevated in BLM1077, indicating that only the latter construct is able to clear SSB from ssDNA. (D) Kinetics of hRad51 nucleoprotein filament clearance by BLM constructs, as monitored by MDCC-SSB fluorescence. dT54 (150 nM) was incubated with 2.8 µM hRad51 for 30 min on ice. BLM constructs were then added at a concentration of 200 nM, and samples were incubated for additional 10 min on ice. Traces labelled BLM1077 (black), BLM1290 (grey) and BLMFL (light grey) were recorded upon rapidly mixing dT54.hRad51.BLM premixtures with 1 µM MDDC-SSB (marked as SSB*) plus 2 mM ATP to monitor hRad51 clearance by BLM. The panel also shows control traces recorded upon mixing MDCC-SSB with buffer alone, MDCC-SSB with free dT54 (in the absence of hRad51 and BLM), and dT54.Rad51.BLM premixtures with MDCC-SSB in the absence of ATP. (In the latter case, traces were very similar in the case of all BLM constructs and also when BLM was omitted.) Traces were corrected for MDCC-SSB fluorescence levels at the start of the reactions. The extent of active hRad51 clearance by BLM, as calculated from the final steady-state MDCC-SSB fluorescence levels, was 41, 41 and 39% in the case of BLM1077, BLM1290 and BLMFL, respectively.

Mentions: We measured the effect of hRad51 on the ssDNA-activated ATPase activity of BLMFL, BLM1290 and BLM1077 (Figure 6A). The hRad51 concentrations required for half-maximal inhibition of the ssDNA-activated BLM ATPase were close to those expected from the stoichiometry of hRad51 binding to ssDNA [3 nt/hRad51 monomer (45)]. Interestingly, the ATPase values at saturating hRad51 concentrations remained ∼20 times higher than the basal ATPase activities of each BLM construct (Figure 6A and C).Figure 6.


Complex activities of the human Bloom's syndrome helicase are encoded in a core region comprising the RecA and Zn-binding domains.

Gyimesi M, Harami GM, Sarlós K, Hazai E, Bikádi Z, Kovács M - Nucleic Acids Res. (2012)

Nucleoprotein filament clearance by BLM constructs. (A) Steady-state ATPase activities of 20 nM BLM1077 (filled squqre), BLM1290 (open circle) and BLMFL (open triangle) measured at 25°C in the presence of 100 nM dT54 and at different hRad51 concentrations. Hill-equation () was used for data fitting in which k0 and kINH are BLM ATPase activities in the absence of hRad51 and in the presence of saturating hRad51 concentration, respectively; cINH is hRad51 concentration; K is the Michaelis constant and n is the cooperativity factor. Obtained values of K and n (not shown) were similar for all three constructs. (B) Steady-state BLM ATPase activities measured as in (A) except using SSB instead of hRad51. SSB tetramer concentrations are indicated. (C) Steady-state ATPase activities of BLM constructs in the absence of DNA (kbasal, black) and in the presence of saturating concentrations of hRad51 and SSB (kINH,hRad51, grey and kINH,SSB, light grey, respectively, cf. panels A and B). Note that kINH,hRad51 was markedly elevated compared to kbasal in all three BLM constructs, indicating hRad51 filament clearance activity. Conversely, kINH,SSB was practically identical to kbasal in BLMFL and BLM1290, but markedly elevated in BLM1077, indicating that only the latter construct is able to clear SSB from ssDNA. (D) Kinetics of hRad51 nucleoprotein filament clearance by BLM constructs, as monitored by MDCC-SSB fluorescence. dT54 (150 nM) was incubated with 2.8 µM hRad51 for 30 min on ice. BLM constructs were then added at a concentration of 200 nM, and samples were incubated for additional 10 min on ice. Traces labelled BLM1077 (black), BLM1290 (grey) and BLMFL (light grey) were recorded upon rapidly mixing dT54.hRad51.BLM premixtures with 1 µM MDDC-SSB (marked as SSB*) plus 2 mM ATP to monitor hRad51 clearance by BLM. The panel also shows control traces recorded upon mixing MDCC-SSB with buffer alone, MDCC-SSB with free dT54 (in the absence of hRad51 and BLM), and dT54.Rad51.BLM premixtures with MDCC-SSB in the absence of ATP. (In the latter case, traces were very similar in the case of all BLM constructs and also when BLM was omitted.) Traces were corrected for MDCC-SSB fluorescence levels at the start of the reactions. The extent of active hRad51 clearance by BLM, as calculated from the final steady-state MDCC-SSB fluorescence levels, was 41, 41 and 39% in the case of BLM1077, BLM1290 and BLMFL, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3351180&req=5

gks008-F6: Nucleoprotein filament clearance by BLM constructs. (A) Steady-state ATPase activities of 20 nM BLM1077 (filled squqre), BLM1290 (open circle) and BLMFL (open triangle) measured at 25°C in the presence of 100 nM dT54 and at different hRad51 concentrations. Hill-equation () was used for data fitting in which k0 and kINH are BLM ATPase activities in the absence of hRad51 and in the presence of saturating hRad51 concentration, respectively; cINH is hRad51 concentration; K is the Michaelis constant and n is the cooperativity factor. Obtained values of K and n (not shown) were similar for all three constructs. (B) Steady-state BLM ATPase activities measured as in (A) except using SSB instead of hRad51. SSB tetramer concentrations are indicated. (C) Steady-state ATPase activities of BLM constructs in the absence of DNA (kbasal, black) and in the presence of saturating concentrations of hRad51 and SSB (kINH,hRad51, grey and kINH,SSB, light grey, respectively, cf. panels A and B). Note that kINH,hRad51 was markedly elevated compared to kbasal in all three BLM constructs, indicating hRad51 filament clearance activity. Conversely, kINH,SSB was practically identical to kbasal in BLMFL and BLM1290, but markedly elevated in BLM1077, indicating that only the latter construct is able to clear SSB from ssDNA. (D) Kinetics of hRad51 nucleoprotein filament clearance by BLM constructs, as monitored by MDCC-SSB fluorescence. dT54 (150 nM) was incubated with 2.8 µM hRad51 for 30 min on ice. BLM constructs were then added at a concentration of 200 nM, and samples were incubated for additional 10 min on ice. Traces labelled BLM1077 (black), BLM1290 (grey) and BLMFL (light grey) were recorded upon rapidly mixing dT54.hRad51.BLM premixtures with 1 µM MDDC-SSB (marked as SSB*) plus 2 mM ATP to monitor hRad51 clearance by BLM. The panel also shows control traces recorded upon mixing MDCC-SSB with buffer alone, MDCC-SSB with free dT54 (in the absence of hRad51 and BLM), and dT54.Rad51.BLM premixtures with MDCC-SSB in the absence of ATP. (In the latter case, traces were very similar in the case of all BLM constructs and also when BLM was omitted.) Traces were corrected for MDCC-SSB fluorescence levels at the start of the reactions. The extent of active hRad51 clearance by BLM, as calculated from the final steady-state MDCC-SSB fluorescence levels, was 41, 41 and 39% in the case of BLM1077, BLM1290 and BLMFL, respectively.
Mentions: We measured the effect of hRad51 on the ssDNA-activated ATPase activity of BLMFL, BLM1290 and BLM1077 (Figure 6A). The hRad51 concentrations required for half-maximal inhibition of the ssDNA-activated BLM ATPase were close to those expected from the stoichiometry of hRad51 binding to ssDNA [3 nt/hRad51 monomer (45)]. Interestingly, the ATPase values at saturating hRad51 concentrations remained ∼20 times higher than the basal ATPase activities of each BLM construct (Figure 6A and C).Figure 6.

Bottom Line: We performed a quantitative mechanistic analysis of truncated BLM constructs that are shorter than the previously identified minimal functional module.Surprisingly, we found that a BLM construct comprising only the two conserved RecA domains and the Zn(2+)-binding domain (residues 642-1077) can efficiently perform all mentioned HR-related activities.The results demonstrate that the Zn(2+)-binding domain is necessary for functional interaction with DNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, ELTE-MTA Momentum Motor Enzymology Research Group, Eötvös University, Pázmány P. s. 1/c, H-1117 Budapest, Hungary.

ABSTRACT
Bloom's syndrome DNA helicase (BLM), a member of the RecQ family, is a key player in homologous recombination (HR)-based error-free DNA repair processes. During HR, BLM exerts various biochemical activities including single-stranded (ss) DNA translocation, separation and annealing of complementary DNA strands, disruption of complex DNA structures (e.g. displacement loops) and contributes to quality control of HR via clearance of Rad51 nucleoprotein filaments. We performed a quantitative mechanistic analysis of truncated BLM constructs that are shorter than the previously identified minimal functional module. Surprisingly, we found that a BLM construct comprising only the two conserved RecA domains and the Zn(2+)-binding domain (residues 642-1077) can efficiently perform all mentioned HR-related activities. The results demonstrate that the Zn(2+)-binding domain is necessary for functional interaction with DNA. We show that the extensions of this core, including the winged-helix domain and the strand separation hairpin identified therein in other RecQ-family helicases, are not required for mechanochemical activity per se and may instead play modulatory roles and mediate protein-protein interactions.

Show MeSH
Related in: MedlinePlus