Point mutations causing Bloom's syndrome abolish ATPase and DNA helicase activities of the BLM protein

Bahr, Anne; Graeve, Fabienne De; Kédinger, Claude; Chatton, Bruno

doi:10.1038/sj.onc.1202389

Cited by 68 publications

(56 citation statements)

References 22 publications

(39 reference statements)

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“…Two disease-causing Bloom missense mutations map to Cys 1036 and Cys 1055 , respectively (32,33), among four conserved cysteine residues in the zinc finger motif. In vitro analysis shows that these mutations abolish BLM ATPase and helicase activities (34). In vitro studies of the RecQ core of the Bloom syndrome protein have shown that when three of the four cysteines in the zinc finger motif were mutated, respectively, the resulting mutants were very unstable.…”

Section: Discussionmentioning

confidence: 99%

The Zinc Finger Motif of Escherichia coli RecQ Is Implicated in Both DNA Binding and Protein Folding

Liu¹,

Rigolet²,

Dou³

et al. 2004

Journal of Biological Chemistry

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The RecQ family of DNA helicases has been shown to be important for the maintenance of genomic integrity. Mutations in human RecQ genes lead to genomic instability and cancer. Several RecQ family of helicases contain a putative zinc finger motif of the C 4 type at the C terminus that has been identified in the crystalline structure of RecQ helicase from Escherichia coli. To better understand the role of this motif in helicase from E. coli, we constructed a series of single mutations altering the conserved cysteines as well as other highly conserved residues. All of the resulting mutant proteins exhibited a high level of susceptibility to degradation, making functional analysis impossible. In contrast, a double mutant protein in which both cysteine residues Cys 397 and Cys 400 in the zinc finger motif were replaced by asparagine residues was purified to homogeneity. Slight local conformational changes were detected, but the rest of the mutant protein has a well defined tertiary structure. Furthermore, the mutant enzyme displayed ATP binding affinity similar to the wild-type enzyme but was severely impaired in DNA binding and in subsequent ATPase and helicase activities. These results revealed that the zinc finger binding motif is involved in maintaining the integrity of the whole protein as well as DNA binding. We also showed that the zinc atom is not essential to enzymatic activity.The transient formation of single-stranded DNA (ssDNA) 1 intermediate is essential to all aspects of DNA metabolism including DNA replication, recombination, and repair. The unwinding and separation of the individual strands of doublestranded DNA (dsDNA) is catalyzed by a class of specialized enzymes known as DNA helicases (1, 2). These enzymes function as molecular motors that use the energy released from the hydrolysis of ATP to unwind and translocate along DNA in a sequential fashion (3-5). These ubiquitous enzymes have been identified in all living organisms from virus to human. It appears that they evolved from a common ancestor (6).The RecQ helicase family is critical to the maintenance of genomic stability in prokaryotes and eukaryotes (7). Mutations of RecQ genes can lead to genomic instability and several human diseases including the Bloom and Werner syndromes (8). Recently, it has been shown that the tumor suppressor BRCA1-associated protein, BACH1, which shares homologies with other members of the RecQ family, possesses ATPase and helicase activities (9). The mutant BACH1 participates directly in breast and ovarian cancer development (9). The RecQ helicase from Escherichia coli is the prototype helicase of this family (10) and has been shown to initiate homologous recombination as well as suppress illegitimate recombination (11, 12).The RecQ helicase family members contain a helicase domain characterized by the presence of seven so-called "helicase" motifs necessary for using energy derived from ATP binding and hydrolysis to unwind DNA (13,14). Sequence analyses revealed that all of the RecQ helicases contain a C-terminal e...

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Section: Discussionmentioning

confidence: 99%

The Zinc Finger Motif of Escherichia coli RecQ Is Implicated in Both DNA Binding and Protein Folding

Liu¹,

Rigolet²,

Dou³

et al. 2004

Journal of Biological Chemistry

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“…The structures of E. coli RecQ and human RECQ1 proteins show that the motif 0 is involved in nucleotide binding, and mutagenesis studies have confirmed that this motif is important for core helicase domain function. For example, substitution of the C-terminal Gln with Ala in RECQ5␤ significantly reduces ATPase activity, and the replacement of the same residue with Arg inactivates the ATPase and helicase activity of murine BLM (Bahr et al, 1998;Garcia et al, 2004). The same mutation is also listed among the naturally occurring mutations of BLM associated with Bloom's syndrome (Ellis et al, 1995).…”

Section: The Core Helicase Domainmentioning

confidence: 99%

RecQ helicases: Multiple structures for multiple functions?

Vindigni

Hickson

2009

HFSP Journal

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“…Chester et al (1998) developed a mouse model for the human Bloom's syndrome, and showed that mouse embryos homozygous for a targeted mutation in the murine Bloom's syndrome gene die by embryonic day 13.5, but little is known about function(s) of BLM. It has been shown that recombinant BLM displays an ATP-and Mg2+ dependent 3' ± 5'-DNA helicase activity (Karow et al, 1997) which is required to correct the genomic instability inherent to BS cells (Bahr et al, 1998;Ne et al, 1999). In the RecQ subfamily, phylogenetic analyses have revealed that the BLM protein is most closely related to Schizosaccharomyces pombe Rqh1 and to Saccharomyces cerevisiae Sgs1p (Murray et al, 1997;Kitao et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Cell cycle regulation of the endogenous wild type Bloom's syndrome DNA helicase

et al. 2000

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Bloom's syndrome (BS) is a rare human autosomal recessive disorder characterized by an increased risk to develop cancer of all types. BS cells are characterized by a generalized genetic instability including a high level of sister chromatid exchanges. BS arises through mutations in both alleles of the BLM gene which encodes a 3' ± 5' DNA helicase identi®ed as a member of the RecQ family. We developed polyclonal antibodies speci®c for the NH 2 -and COOH-terminal region of BLM. Using these antibodies, we analysed BLM expression during the cell cycle and showed that the BLM protein accumulates to high levels in S phase, persists in G2/ M and sharply declines in G1, strongly suggestive of degradation during mitosis. The BLM protein is subject to post-translational modi®cations in mitosis, as revealed by slow migrating forms of BLM found in both demecolcine-treated cells and in mitotic cells isolated from non-treated asynchronous populations. Phosphatase treatment indicated that phosphorylation events were solely responsible for the appearance of the retarded moieties, a possible signal for subsequent degradation. Together, these results are consistent with a role of BLM in a replicative (S phase) and/or post-replicative (G2 phase) process.

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Point mutations causing Bloom's syndrome abolish ATPase and DNA helicase activities of the BLM protein

Cited by 68 publications

References 22 publications

The Zinc Finger Motif of Escherichia coli RecQ Is Implicated in Both DNA Binding and Protein Folding

The Zinc Finger Motif of Escherichia coli RecQ Is Implicated in Both DNA Binding and Protein Folding

RecQ helicases: Multiple structures for multiple functions?

Cell cycle regulation of the endogenous wild type Bloom's syndrome DNA helicase

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