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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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Related in: MedlinePlus

BLM1077 performs effective dsDNA unwinding and detectable strand annealing. (A) DNA unwinding reactions using fluorescein-labelled (asterisks) splayed-arm DNA substrate containing 33 bp of dsDNA and 21–21 nt ssDNA regions. BLM1077 and BLM1290 were incubated with the DNA substrate at different concentrations (0–1 µM) in the presence of 2 mM ATP for 15 min at 37°C. Samples were then deproteinised and run on 12% acrylamide gel. (B) Dependence of the extent of unwinding (%) on BLM1077 (filled square) and BLM1290 (○) concentration. Data were corrected for the fraction of ssDNA in the absence of protein. (C–F) Strand annealing reactions using unlabelled and fluorescein-labelled (asterisks) ssDNA substrates in the absence of nucleotides (‘–Nu’, C and D) and in the presence of 2 mM AMPPNP (E and F). BLM1077 (filled square) and BLM1290 (○) (0–1 µM) were incubated with the two ssDNA strands capable of forming a splayed-arm structure (identical to those in panel A) for 15 min at 37°C. Samples were then deproteinised and run on 12% acrylamide gel. Data were corrected for the fraction of dsDNA in the absence of protein.
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gks008-F4: BLM1077 performs effective dsDNA unwinding and detectable strand annealing. (A) DNA unwinding reactions using fluorescein-labelled (asterisks) splayed-arm DNA substrate containing 33 bp of dsDNA and 21–21 nt ssDNA regions. BLM1077 and BLM1290 were incubated with the DNA substrate at different concentrations (0–1 µM) in the presence of 2 mM ATP for 15 min at 37°C. Samples were then deproteinised and run on 12% acrylamide gel. (B) Dependence of the extent of unwinding (%) on BLM1077 (filled square) and BLM1290 (○) concentration. Data were corrected for the fraction of ssDNA in the absence of protein. (C–F) Strand annealing reactions using unlabelled and fluorescein-labelled (asterisks) ssDNA substrates in the absence of nucleotides (‘–Nu’, C and D) and in the presence of 2 mM AMPPNP (E and F). BLM1077 (filled square) and BLM1290 (○) (0–1 µM) were incubated with the two ssDNA strands capable of forming a splayed-arm structure (identical to those in panel A) for 15 min at 37°C. Samples were then deproteinised and run on 12% acrylamide gel. Data were corrected for the fraction of dsDNA in the absence of protein.

Mentions: The above experiments with BLM1077 demonstrated that the truncation of the N-terminal, WH, HRDC and C-terminal domains did not markedly affect the ATPase and ssDNA translocation properties of BLM. Next, we tested the DNA strand separation activity of BLM1077 on a splayed-arm DNA substrate (Figure 4A and B). Despite the fact that the WH domain [harbouring the WH-pin implicated in strand separation by RecQ1 (29,32) and WRN (31)] is entirely missing from BLM1077, surprisingly we found the unwinding profile of BLM1077 to be similar or even slightly enhanced compared to that of BLM1290 (Figure 4A and B). These data demonstrate that the WH domain is not necessary for dsDNA strand separation by BLM.Figure 4.


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 performs effective dsDNA unwinding and detectable strand annealing. (A) DNA unwinding reactions using fluorescein-labelled (asterisks) splayed-arm DNA substrate containing 33 bp of dsDNA and 21–21 nt ssDNA regions. BLM1077 and BLM1290 were incubated with the DNA substrate at different concentrations (0–1 µM) in the presence of 2 mM ATP for 15 min at 37°C. Samples were then deproteinised and run on 12% acrylamide gel. (B) Dependence of the extent of unwinding (%) on BLM1077 (filled square) and BLM1290 (○) concentration. Data were corrected for the fraction of ssDNA in the absence of protein. (C–F) Strand annealing reactions using unlabelled and fluorescein-labelled (asterisks) ssDNA substrates in the absence of nucleotides (‘–Nu’, C and D) and in the presence of 2 mM AMPPNP (E and F). BLM1077 (filled square) and BLM1290 (○) (0–1 µM) were incubated with the two ssDNA strands capable of forming a splayed-arm structure (identical to those in panel A) for 15 min at 37°C. Samples were then deproteinised and run on 12% acrylamide gel. Data were corrected for the fraction of dsDNA in the absence of protein.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351180&req=5

gks008-F4: BLM1077 performs effective dsDNA unwinding and detectable strand annealing. (A) DNA unwinding reactions using fluorescein-labelled (asterisks) splayed-arm DNA substrate containing 33 bp of dsDNA and 21–21 nt ssDNA regions. BLM1077 and BLM1290 were incubated with the DNA substrate at different concentrations (0–1 µM) in the presence of 2 mM ATP for 15 min at 37°C. Samples were then deproteinised and run on 12% acrylamide gel. (B) Dependence of the extent of unwinding (%) on BLM1077 (filled square) and BLM1290 (○) concentration. Data were corrected for the fraction of ssDNA in the absence of protein. (C–F) Strand annealing reactions using unlabelled and fluorescein-labelled (asterisks) ssDNA substrates in the absence of nucleotides (‘–Nu’, C and D) and in the presence of 2 mM AMPPNP (E and F). BLM1077 (filled square) and BLM1290 (○) (0–1 µM) were incubated with the two ssDNA strands capable of forming a splayed-arm structure (identical to those in panel A) for 15 min at 37°C. Samples were then deproteinised and run on 12% acrylamide gel. Data were corrected for the fraction of dsDNA in the absence of protein.
Mentions: The above experiments with BLM1077 demonstrated that the truncation of the N-terminal, WH, HRDC and C-terminal domains did not markedly affect the ATPase and ssDNA translocation properties of BLM. Next, we tested the DNA strand separation activity of BLM1077 on a splayed-arm DNA substrate (Figure 4A and B). Despite the fact that the WH domain [harbouring the WH-pin implicated in strand separation by RecQ1 (29,32) and WRN (31)] is entirely missing from BLM1077, surprisingly we found the unwinding profile of BLM1077 to be similar or even slightly enhanced compared to that of BLM1290 (Figure 4A and B). These data demonstrate that the WH domain is not necessary for dsDNA strand separation by BLM.Figure 4.

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