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Janscak, P, Garcia, PL, Hamburger, F, Makuta, Y, Shiraishi, K, Imai, Y, Ikeda, H and Bickle, TA (2003) Characterization and mutational analysis of the RecQ core of the bloom syndrome protein. J. Mol. Biol. 330:29-42
Bloom syndrome protein forms an oligomeric ring structure and belongs to a group of DNA helicases showing extensive homology to the Escherichia coli DNA helicase RecQ, a suppressor of illegitimate recombination. After over-production in E.coli, we have purified the RecQ core of BLM consisting of the DEAH, RecQ-Ct and HRDC domains (amino acid residues 642-1290). The BLM(642-1290) fragment could function as a DNA-stimulated ATPase and as a DNA helicase, displaying the same substrate specificity as the full-size protein. Gel-filtration experiments revealed that BLM(642-1290) exists as a monomer both in solution and in its single-stranded DNA-bound form, even in the presence of Mg(2+) and ATPgammaS. Rates of ATP hydrolysis and DNA unwinding by BLM(642-1290) showed a hyperbolic dependence on ATP concentration, excluding a co-operative interaction between ATP-binding sites. Using a lambda Spi(-) assay, we have found that the BLM(642-1290) fragment is able to partially substitute for the RecQ helicase in suppressing illegitimate recombination in E.coli. A deletion of 182 C-terminal amino acid residues of BLM(642-1290), including the HRDC domain, resulted in helicase and single-stranded DNA-binding defects, whereas kinetic parameters for ATP hydrolysis of this mutant were close to the BLM(642-1290) values. This confirms the prediction that the HRDC domain serves as an auxiliary DNA-binding domain. Mutations at several conserved residues within the RecQ-Ct domain of BLM reduced ATPase and helicase activities severely as well as single-stranded DNA-binding of the enzyme. Together, these data define a minimal helicase domain of BLM and demonstrate its ability to act as a suppressor of illegitimate recombination.
Adenosine Triphosphatases/chemistry; Adenosine Triphosphatases/genetics; Adenosine Triphosphatases/isolation & purification; Adenosine Triphosphatases/metabolism; Adenosine Triphosphate/analogs & derivatives; Adenosine Triphosphate/metabolism; Amino Acid Sequence; Biochemistry/methods; DNA Helicases/chemistry; DNA Helicases/genetics; DNA Helicases/isolation & purification; DNA Helicases/metabolism; Escherichia coli/genetics; Humans; Magnesium/metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Protein Structure, Tertiary; RecQ Helicases; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Recombination, Genetic; Sequence Deletion; Sequence Homology, Amino Acid; Solutions; Structure-Activity Relationship; Substrate Specificity