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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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BLM1077 and BLM1290 translocate along ssDNA with similar mechanochemical coupling and processivity. (A) Transient kinetics of Pi liberation from ATP by BLM1077 during single-round ssDNA translocation. BLM1077 (0.1 µM) plus oligo-dT [1 µM, lengths (bottom to top) were 12, 15, 18, 23, 30, 45, 63 and 72 nt] was mixed with 0.5 mM ATP plus 8 mg/ml heparin (used as a protein trap) in the stopped-flow apparatus. Pi release was monitored using MDCC-PBP fluorescence (3 µM in all syringes). Fluorescence changes were converted to Pi concentration by using calibration curves. As previously described for BLM1290 (41,51), traces at oligo-dT lengths >12 nt consisted of an exponential burst with an amplitude of 1 mol Pi/mol BLM1077 (Supplementary Figure S4A), and two additional phases reflecting ATP hydrolysis during ssDNA translocation and in the trap-bound state, respectively. (B) Oligo-dT length dependence of the amplitudes of Pi production during the translocation phase. Data were fitted using the equation described for BLM1290 (41). The fit yielded translocation parameters for BLM1077 similar to those of BLM1290 (Table 1). (C) Transient kinetics of Pi liberation upon mixing 0.05 µM BLM1077 (black line) or BLM1290 (grey line) plus 90 µM (nt concentration) m13mp18 phage circular ssDNA with 0.5 mM ATP plus 8 mg/ml heparin in the stopped-flow apparatus. The amplitudes of Pi production during the translocation phase indicated similar mean run lengths (‹m›) and processivities (P) for the two constructs at the applied heparin concentration (‹m›BLM1077 = 21 nt, PBLM1077 = 0.955; ‹m›BLM1290 = 24 nt, PBLM1290 = 0.961).
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gks008-F3: BLM1077 and BLM1290 translocate along ssDNA with similar mechanochemical coupling and processivity. (A) Transient kinetics of Pi liberation from ATP by BLM1077 during single-round ssDNA translocation. BLM1077 (0.1 µM) plus oligo-dT [1 µM, lengths (bottom to top) were 12, 15, 18, 23, 30, 45, 63 and 72 nt] was mixed with 0.5 mM ATP plus 8 mg/ml heparin (used as a protein trap) in the stopped-flow apparatus. Pi release was monitored using MDCC-PBP fluorescence (3 µM in all syringes). Fluorescence changes were converted to Pi concentration by using calibration curves. As previously described for BLM1290 (41,51), traces at oligo-dT lengths >12 nt consisted of an exponential burst with an amplitude of 1 mol Pi/mol BLM1077 (Supplementary Figure S4A), and two additional phases reflecting ATP hydrolysis during ssDNA translocation and in the trap-bound state, respectively. (B) Oligo-dT length dependence of the amplitudes of Pi production during the translocation phase. Data were fitted using the equation described for BLM1290 (41). The fit yielded translocation parameters for BLM1077 similar to those of BLM1290 (Table 1). (C) Transient kinetics of Pi liberation upon mixing 0.05 µM BLM1077 (black line) or BLM1290 (grey line) plus 90 µM (nt concentration) m13mp18 phage circular ssDNA with 0.5 mM ATP plus 8 mg/ml heparin in the stopped-flow apparatus. The amplitudes of Pi production during the translocation phase indicated similar mean run lengths (‹m›) and processivities (P) for the two constructs at the applied heparin concentration (‹m›BLM1077 = 21 nt, PBLM1077 = 0.955; ‹m›BLM1290 = 24 nt, PBLM1290 = 0.961).

Mentions: To determine the translocation processivity of BLM1077, we measured the kinetics of Pi production form ATP in single-round translocation conditions in the presence of oligo-dT substrates of different length (Figure 3). Heparin was used as a protein trap to achieve single-round conditions. Heparin turned out to be an efficient trap for BLM1077, because it blocked the rebinding of DNA to the dissociated helicase, while it did not significantly enhance its DNA-free ATPase activity (Figure 3 and Supplementary Figure S4). As we described earlier for BLM1290 (41), upon mixing BLM1077 plus oligo-dT substrate with ATP plus heparin, the experimental traces showed multiphasic profiles. A first pre-steady-state exponential burst (Supplementary Figure S4) was followed by two other distinct phases corresponding to ATPase cycling during translocation along DNA and in the dissociated (trap-bound) state of the enzyme, respectively. The existence of a pre-steady-state exponential burst corresponding to a single ATP turnover by BLM1077 [identical to that by BLM1290 (41)] suggested that the rate-limiting step in the ATPase cycle during translocation was not changed by the truncation. The ATPase rate during the translocation-based phase was in good agreement with that determined using the PK/LDH-coupled ATPase assay (Figures 2 and 3; Supplementary Figure S4 and Table 1). The DNA length dependence of the amplitude of Pi production during translocation can be utilized to determine the mechanochemical coupling stoichiometry (C, ATP consumed/nt translocated) and processivity (P) of DNA-based motor proteins (41,51,52). Applying our previously published model (51) to the data sets showed that the coupling stoichiometry, C = 0.83 ± 0.11 ATP/nt, was practically identical to that determined earlier for BLM1290 (Figure 3B and Table 1) (41). This value reflects that the mean step size of ∼1 nt/ATP was not altered by the truncation. The processivity values of BLM1077 at the applied heparin concentrations (P = 0.956 and 0.951 at 6 and 8 mg/ml heparin, respectively; indicating a mean of 22 and 19 cycles taken in a single run) suggested that BLM1077 translocates slightly more processively along ssDNA than that reported for BLM1290 in similar conditions (41). To directly compare the processivities of BLM1077 and BLM1290, we performed single-round translocation experiments with both constructs in the presence of circular m13 phage ssDNA and 8 mg/ml heparin (Figure 3C). These experiments resulted in similar processivities for the two constructs, with a mean of 21 and 24 steps in a single run performed by BLM1077 and BLM1290, respectively.Figure 3.


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)

BLM1077 and BLM1290 translocate along ssDNA with similar mechanochemical coupling and processivity. (A) Transient kinetics of Pi liberation from ATP by BLM1077 during single-round ssDNA translocation. BLM1077 (0.1 µM) plus oligo-dT [1 µM, lengths (bottom to top) were 12, 15, 18, 23, 30, 45, 63 and 72 nt] was mixed with 0.5 mM ATP plus 8 mg/ml heparin (used as a protein trap) in the stopped-flow apparatus. Pi release was monitored using MDCC-PBP fluorescence (3 µM in all syringes). Fluorescence changes were converted to Pi concentration by using calibration curves. As previously described for BLM1290 (41,51), traces at oligo-dT lengths >12 nt consisted of an exponential burst with an amplitude of 1 mol Pi/mol BLM1077 (Supplementary Figure S4A), and two additional phases reflecting ATP hydrolysis during ssDNA translocation and in the trap-bound state, respectively. (B) Oligo-dT length dependence of the amplitudes of Pi production during the translocation phase. Data were fitted using the equation described for BLM1290 (41). The fit yielded translocation parameters for BLM1077 similar to those of BLM1290 (Table 1). (C) Transient kinetics of Pi liberation upon mixing 0.05 µM BLM1077 (black line) or BLM1290 (grey line) plus 90 µM (nt concentration) m13mp18 phage circular ssDNA with 0.5 mM ATP plus 8 mg/ml heparin in the stopped-flow apparatus. The amplitudes of Pi production during the translocation phase indicated similar mean run lengths (‹m›) and processivities (P) for the two constructs at the applied heparin concentration (‹m›BLM1077 = 21 nt, PBLM1077 = 0.955; ‹m›BLM1290 = 24 nt, PBLM1290 = 0.961).
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gks008-F3: BLM1077 and BLM1290 translocate along ssDNA with similar mechanochemical coupling and processivity. (A) Transient kinetics of Pi liberation from ATP by BLM1077 during single-round ssDNA translocation. BLM1077 (0.1 µM) plus oligo-dT [1 µM, lengths (bottom to top) were 12, 15, 18, 23, 30, 45, 63 and 72 nt] was mixed with 0.5 mM ATP plus 8 mg/ml heparin (used as a protein trap) in the stopped-flow apparatus. Pi release was monitored using MDCC-PBP fluorescence (3 µM in all syringes). Fluorescence changes were converted to Pi concentration by using calibration curves. As previously described for BLM1290 (41,51), traces at oligo-dT lengths >12 nt consisted of an exponential burst with an amplitude of 1 mol Pi/mol BLM1077 (Supplementary Figure S4A), and two additional phases reflecting ATP hydrolysis during ssDNA translocation and in the trap-bound state, respectively. (B) Oligo-dT length dependence of the amplitudes of Pi production during the translocation phase. Data were fitted using the equation described for BLM1290 (41). The fit yielded translocation parameters for BLM1077 similar to those of BLM1290 (Table 1). (C) Transient kinetics of Pi liberation upon mixing 0.05 µM BLM1077 (black line) or BLM1290 (grey line) plus 90 µM (nt concentration) m13mp18 phage circular ssDNA with 0.5 mM ATP plus 8 mg/ml heparin in the stopped-flow apparatus. The amplitudes of Pi production during the translocation phase indicated similar mean run lengths (‹m›) and processivities (P) for the two constructs at the applied heparin concentration (‹m›BLM1077 = 21 nt, PBLM1077 = 0.955; ‹m›BLM1290 = 24 nt, PBLM1290 = 0.961).
Mentions: To determine the translocation processivity of BLM1077, we measured the kinetics of Pi production form ATP in single-round translocation conditions in the presence of oligo-dT substrates of different length (Figure 3). Heparin was used as a protein trap to achieve single-round conditions. Heparin turned out to be an efficient trap for BLM1077, because it blocked the rebinding of DNA to the dissociated helicase, while it did not significantly enhance its DNA-free ATPase activity (Figure 3 and Supplementary Figure S4). As we described earlier for BLM1290 (41), upon mixing BLM1077 plus oligo-dT substrate with ATP plus heparin, the experimental traces showed multiphasic profiles. A first pre-steady-state exponential burst (Supplementary Figure S4) was followed by two other distinct phases corresponding to ATPase cycling during translocation along DNA and in the dissociated (trap-bound) state of the enzyme, respectively. The existence of a pre-steady-state exponential burst corresponding to a single ATP turnover by BLM1077 [identical to that by BLM1290 (41)] suggested that the rate-limiting step in the ATPase cycle during translocation was not changed by the truncation. The ATPase rate during the translocation-based phase was in good agreement with that determined using the PK/LDH-coupled ATPase assay (Figures 2 and 3; Supplementary Figure S4 and Table 1). The DNA length dependence of the amplitude of Pi production during translocation can be utilized to determine the mechanochemical coupling stoichiometry (C, ATP consumed/nt translocated) and processivity (P) of DNA-based motor proteins (41,51,52). Applying our previously published model (51) to the data sets showed that the coupling stoichiometry, C = 0.83 ± 0.11 ATP/nt, was practically identical to that determined earlier for BLM1290 (Figure 3B and Table 1) (41). This value reflects that the mean step size of ∼1 nt/ATP was not altered by the truncation. The processivity values of BLM1077 at the applied heparin concentrations (P = 0.956 and 0.951 at 6 and 8 mg/ml heparin, respectively; indicating a mean of 22 and 19 cycles taken in a single run) suggested that BLM1077 translocates slightly more processively along ssDNA than that reported for BLM1290 in similar conditions (41). To directly compare the processivities of BLM1077 and BLM1290, we performed single-round translocation experiments with both constructs in the presence of circular m13 phage ssDNA and 8 mg/ml heparin (Figure 3C). These experiments resulted in similar processivities for the two constructs, with a mean of 21 and 24 steps in a single run performed by BLM1077 and BLM1290, respectively.Figure 3.

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