Structural and functional characterizations reveal the importance of a zinc binding domain in Bloom's syndrome helicase.
Bloom's syndrome (BS) is an autosomal recessive human disorder characterized by genomic instability and a predisposition to a wide variety of cancers. The gene mutated in BS, BLM, encodes a protein containing three domains: an N-terminal domain whose function remains elusive, a helicase domain characterized by seven 'signature' motifs conserved in a wide range of helicases and a C-terminal extension that can be further divided into two sub-domains: RecQ-Ct and HRDC. The RecQ-Ct domain appears essential because two point-mutations altering highly conserved cysteine residues within this domain have been found in BS patients. We report herein that BLM contains a zinc ion. Modelling studies suggest that four conserved cysteine residues within the RecQ-Ct domain coordinate this zinc ion and subsequent mutagenesis studies further confirm this prediction. Biochemical and biophysical studies show that the ATPase, helicase and DNA binding activities of the mutants are severely modified. Structural analysis of both wild-type and mutant proteins reveal that alteration of cysteine residues does not significantly change the overall conformation. The observed defects in ATPase and helicase activities were inferred to result from a compromise of DNA binding. Our results implicate an important role of this zinc binding domain in both DNA binding and protein conformation. They could be pivotal for understanding the molecular basis of BS disease.
Guo
Rong-bin
RB
Laboratoire de Biotechnologies et Pharmacologie Génétique Appliquée CNRS UMR 8113, Ecole Normale Supérieure (ENS) Cachan 61 avenue du Président Wilson, 94235 Cachan cedex, France.
Rigolet
Pascal
P
Zargarian
Loussiné
L
Fermandjian
Serge
S
Xi
Xu Guang
XG
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry; D004795:Enzyme Stability; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary; D053484:RecQ Helicases; D016415:Sequence Alignment; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C097151:Bloom syndrome protein; D004265:DNA Helicases; D053484:RecQ Helicases; D015032:Zinc; D003545:Cysteine