title: Characterization and mutational analysis of the RecQ core of the bloom syndrome protein.
abstract: 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.
authors.affiliation: Division of Molecular Microbiology, Biozentrum, University of Basel, Klingelbergstr. 50-70, Switzerland. pjanscak@imr.unizh.ch
authors.forename: Pavel
authors.initials: P
authors.lastname: Janscak
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Garcia
authors.forename: Fabienne
authors.initials: F
authors.lastname: Hamburger
authors.forename: Yoko
authors.initials: Y
authors.lastname: Makuta
authors.forename: Kouya
authors.initials: K
authors.lastname: Shiraishi
authors.forename: Yukiho
authors.initials: Y
authors.lastname: Imai
authors.forename: Hideo
authors.initials: H
authors.lastname: Ikeda
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Bickle
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D001671:Biochemistry / Q000379:methods; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D053484:RecQ Helicases; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D012996:Solutions; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C097151:Bloom syndrome protein; C506725:RECQL protein, human; C506724:RecQ protein, E coli; D004265:DNA Helicases; D053484:RecQ Helicases; D008274:Magnesium