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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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Homology model-based representation of the BLM segment comprising the RecA, ZnBD and WH domains. Shown are homology-modelled structures of BLM in two different conformations, determined crystallographically for EcRecQ (1OYY, A) and human RecQ1 (2WWY, B). Protein segments present in BLM1005 (comprising the two RecA domains) are shown in blue, whereas the additional segment present in BLM1077 (including the ZnBD) is shown in yellow. The green segment (WH domain) is missing from both BLM1005 and BLM1077, but is present in BLM1290 and BLMFL. Pin-like elements implicated in DNA strand separation in various helicases are shown in red. The bound DNA present in the 2WWY-based model is shown in grey.
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gks008-F1: Homology model-based representation of the BLM segment comprising the RecA, ZnBD and WH domains. Shown are homology-modelled structures of BLM in two different conformations, determined crystallographically for EcRecQ (1OYY, A) and human RecQ1 (2WWY, B). Protein segments present in BLM1005 (comprising the two RecA domains) are shown in blue, whereas the additional segment present in BLM1077 (including the ZnBD) is shown in yellow. The green segment (WH domain) is missing from both BLM1005 and BLM1077, but is present in BLM1290 and BLMFL. Pin-like elements implicated in DNA strand separation in various helicases are shown in red. The bound DNA present in the 2WWY-based model is shown in grey.

Mentions: To identify the structural elements that are essential for the various mechanochemical activities of BLM, in the present study we compared the mechanistic properties of the previously investigated BLMFL and BLM1290 constructs with those of new constructs that are shorter than previously characterized ones. These include BLM amino acids 642–1005 (referred to as BLM1005) comprising solely the two RecA-core domains, and BLM amino acids 642–1077 (BLM1077) consisting of the RecA cores and the ZnBD (Figure 1). Surprisingly, we found that BLM1077 retains all of the ATPase, ssDNA translocation and dsDNA unwinding activities, exhibiting highly similar mechanistic parameters to those of BLM1290. The results demonstrate that the RecA domains and the ZnBD comprise a minimal functional helicase core even in the absence of the WH domain that was proposed to be the strand-separating unit of other RecQ helicases (29,31,32). In addition, BLM1077 exhibits more pronounced strand annealing than BLM1290, which shows that previously implicated C-terminal regions are not essential for this activity. We also demonstrate that BLM1077 is capable of disrupting D-loop structures and it has an enhanced capability for nucleoprotein disassembly.Figure 1.


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)

Homology model-based representation of the BLM segment comprising the RecA, ZnBD and WH domains. Shown are homology-modelled structures of BLM in two different conformations, determined crystallographically for EcRecQ (1OYY, A) and human RecQ1 (2WWY, B). Protein segments present in BLM1005 (comprising the two RecA domains) are shown in blue, whereas the additional segment present in BLM1077 (including the ZnBD) is shown in yellow. The green segment (WH domain) is missing from both BLM1005 and BLM1077, but is present in BLM1290 and BLMFL. Pin-like elements implicated in DNA strand separation in various helicases are shown in red. The bound DNA present in the 2WWY-based model is shown in grey.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks008-F1: Homology model-based representation of the BLM segment comprising the RecA, ZnBD and WH domains. Shown are homology-modelled structures of BLM in two different conformations, determined crystallographically for EcRecQ (1OYY, A) and human RecQ1 (2WWY, B). Protein segments present in BLM1005 (comprising the two RecA domains) are shown in blue, whereas the additional segment present in BLM1077 (including the ZnBD) is shown in yellow. The green segment (WH domain) is missing from both BLM1005 and BLM1077, but is present in BLM1290 and BLMFL. Pin-like elements implicated in DNA strand separation in various helicases are shown in red. The bound DNA present in the 2WWY-based model is shown in grey.
Mentions: To identify the structural elements that are essential for the various mechanochemical activities of BLM, in the present study we compared the mechanistic properties of the previously investigated BLMFL and BLM1290 constructs with those of new constructs that are shorter than previously characterized ones. These include BLM amino acids 642–1005 (referred to as BLM1005) comprising solely the two RecA-core domains, and BLM amino acids 642–1077 (BLM1077) consisting of the RecA cores and the ZnBD (Figure 1). Surprisingly, we found that BLM1077 retains all of the ATPase, ssDNA translocation and dsDNA unwinding activities, exhibiting highly similar mechanistic parameters to those of BLM1290. The results demonstrate that the RecA domains and the ZnBD comprise a minimal functional helicase core even in the absence of the WH domain that was proposed to be the strand-separating unit of other RecQ helicases (29,31,32). In addition, BLM1077 exhibits more pronounced strand annealing than BLM1290, which shows that previously implicated C-terminal regions are not essential for this activity. We also demonstrate that BLM1077 is capable of disrupting D-loop structures and it has an enhanced capability for nucleoprotein disassembly.Figure 1.

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