Characterization and Mutational Analysis of the RecQ Core of the Bloom Syndrome Protein

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 BLM642–1290 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 BLM642–1290 exists as a monomer both in solution and in its single-stranded DNA-bound form, even in the presence of Mg2+ and ATPγS. Rates of ATP hydrolysis and DNA unwinding by BLM642–1290 showed a hyperbolic dependence on ATP concentration, excluding a co-operative interaction between ATP-binding sites. Using a λ Spi− assay, we have found that the BLM642–1290 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 BLM642–1290, 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 BLM642–1290 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.