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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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DNA length-dependent ATPase kinetics indicate active ssDNA translocation by BLM1077. Shown are steady-state ATPase kcat (filled square) and KDNA (open square, ssDNA concentration required for half-maximal activation) values of 20 nM BLM1077 in the presence of oligo-dT substrates of different length at 25°C. kcat values were fitted (solid line) with the equation described previously for BLM1290 (41). Determined mechanistic parameters are listed in Table 1. The break point in KDNA values indicated a binding site size (b) of ∼10 nt, which is detectably smaller than that previously described for BLM1290 (∼14 nt) (41). See Supplementary Figure S3 for comparison of ATPase profiles of BLM1077 with those of BLM1290 and BLMFL in the presence of DNA substrates of different structure. Error bars represent SEM in all figures.
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gks008-F2: DNA length-dependent ATPase kinetics indicate active ssDNA translocation by BLM1077. Shown are steady-state ATPase kcat (filled square) and KDNA (open square, ssDNA concentration required for half-maximal activation) values of 20 nM BLM1077 in the presence of oligo-dT substrates of different length at 25°C. kcat values were fitted (solid line) with the equation described previously for BLM1290 (41). Determined mechanistic parameters are listed in Table 1. The break point in KDNA values indicated a binding site size (b) of ∼10 nt, which is detectably smaller than that previously described for BLM1290 (∼14 nt) (41). See Supplementary Figure S3 for comparison of ATPase profiles of BLM1077 with those of BLM1290 and BLMFL in the presence of DNA substrates of different structure. Error bars represent SEM in all figures.

Mentions: To test the ssDNA translocation activity of BLM1077, we measured the dependence of its steady-state ATPase kcat on oligo-dT substrate length (Figure 2; lengths expressed in nucleotide units). Similarly to our earlier findings on BLM1290 (41), BLM1077 showed two characteristic features: the steady-state kcat values increased and showed saturation with increasing DNA length, whereas the KDNA values showed a drastic length dependence <10 nt [delineating the binding site size (b) of the helicase], and stagnated above this length. Analysis of the data confirmed that BLM1077 actively translocates along the ssDNA track, with slightly higher ATPase rates both during translocation (ktrans) and at the 5′-end (kend) than BLM1290 (Table 1) (41). Dissociation from the 5′-end (koff,end) was also accelerated compared to BLM1290, resulting in saturation of kcat values at shorter DNA lengths. The data also indicated that the step size (s) remained 1 nt/ATP. However, this parameter was less robust due to the relatively small difference between ktrans and kend and the high koff,end value.Figure 2.


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)

DNA length-dependent ATPase kinetics indicate active ssDNA translocation by BLM1077. Shown are steady-state ATPase kcat (filled square) and KDNA (open square, ssDNA concentration required for half-maximal activation) values of 20 nM BLM1077 in the presence of oligo-dT substrates of different length at 25°C. kcat values were fitted (solid line) with the equation described previously for BLM1290 (41). Determined mechanistic parameters are listed in Table 1. The break point in KDNA values indicated a binding site size (b) of ∼10 nt, which is detectably smaller than that previously described for BLM1290 (∼14 nt) (41). See Supplementary Figure S3 for comparison of ATPase profiles of BLM1077 with those of BLM1290 and BLMFL in the presence of DNA substrates of different structure. Error bars represent SEM in all figures.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks008-F2: DNA length-dependent ATPase kinetics indicate active ssDNA translocation by BLM1077. Shown are steady-state ATPase kcat (filled square) and KDNA (open square, ssDNA concentration required for half-maximal activation) values of 20 nM BLM1077 in the presence of oligo-dT substrates of different length at 25°C. kcat values were fitted (solid line) with the equation described previously for BLM1290 (41). Determined mechanistic parameters are listed in Table 1. The break point in KDNA values indicated a binding site size (b) of ∼10 nt, which is detectably smaller than that previously described for BLM1290 (∼14 nt) (41). See Supplementary Figure S3 for comparison of ATPase profiles of BLM1077 with those of BLM1290 and BLMFL in the presence of DNA substrates of different structure. Error bars represent SEM in all figures.
Mentions: To test the ssDNA translocation activity of BLM1077, we measured the dependence of its steady-state ATPase kcat on oligo-dT substrate length (Figure 2; lengths expressed in nucleotide units). Similarly to our earlier findings on BLM1290 (41), BLM1077 showed two characteristic features: the steady-state kcat values increased and showed saturation with increasing DNA length, whereas the KDNA values showed a drastic length dependence <10 nt [delineating the binding site size (b) of the helicase], and stagnated above this length. Analysis of the data confirmed that BLM1077 actively translocates along the ssDNA track, with slightly higher ATPase rates both during translocation (ktrans) and at the 5′-end (kend) than BLM1290 (Table 1) (41). Dissociation from the 5′-end (koff,end) was also accelerated compared to BLM1290, resulting in saturation of kcat values at shorter DNA lengths. The data also indicated that the step size (s) remained 1 nt/ATP. However, this parameter was less robust due to the relatively small difference between ktrans and kend and the high koff,end value.Figure 2.

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