Selective Cleavage of BLM, the Bloom Syndrome Protein, during Apoptotic Cell Death*

Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RECQ-like helicase that is presumed to function in mammalian DNA replication, recombination, or repair. We show here that BLM, but not the related RECQ-like helicase WRN, is rapidly cleaved in cells undergoing apoptosis. BLM was cleaved to 47- and 110-kDa major fragments, with kinetics similar to the apoptotic cleavage of poly(A)DP-ribose polymerase. BLM cleavage was prevented by a caspase 3 inhibitor and did not occur in caspase 3-deficient cells. Moreover, recombinant BLM was cleaved to 47- and 110-kDa fragments by caspase 3, but not caspase 6,in vitro. The caspase 3 recognition sequence412TEVD415 was verified by mutating aspartate 415 to glycine and showing that this mutation rendered BLM resistant to caspase 3 cleavage. Cleavage did not abolish the BLM helicase activity but abolished BLM nuclear foci and the association of BLM with condensed DNA and the insoluble matrix. The results suggest that BLM, but not WRN, is an early selected target during the execution of apoptosis.

[1]  N. Ellis,et al.  Characterization of the human and mouse WRN 3'-->5' exonuclease. , 2000, Nucleic acids research.

[2]  C. Jaulin,et al.  Cell cycle regulation of the endogenous wild type Bloom's syndrome DNA helicase , 2000, Oncogene.

[3]  S. Elledge,et al.  BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. , 2000, Genes & development.

[4]  D. Ramsden,et al.  Ku complex interacts with and stimulates the Werner protein. , 2000, Genes & development.

[5]  S. Jackson,et al.  Expression and nuclear localization of BLM, a chromosome stability protein mutated in Bloom's syndrome, suggest a role in recombination during meiotic prophase. , 2000, Journal of cell science.

[6]  I. Hickson,et al.  RecQ family helicases: roles in cancer and aging. , 2000, Current opinion in genetics & development.

[7]  P. Leder,et al.  The Werner Syndrome Gene Product Co-purifies with the DNA Replication Complex and Interacts with PCNA and Topoisomerase I* , 1999, The Journal of Biological Chemistry.

[8]  P. Pandolfi,et al.  A role for PML and the nuclear body in genomic stability , 1999, Oncogene.

[9]  H. Youssoufian,et al.  Localization of the bloom syndrome helicase to punctate nuclear structures and the nuclear matrix and regulation during the cell cycle: Comparison with the werner's syndrome helicase , 1999, Molecular carcinogenesis.

[10]  N. Ellis,et al.  Purification of overexpressed hexahistidine-tagged BLM N431 as oligomeric complexes. , 1999, Protein expression and purification.

[11]  E. Yeh,et al.  Pml Is Critical for Nd10 Formation and Recruits the Pml-Interacting Protein Daxx to This Nuclear Structure When Modified by Sumo-1 , 1999, The Journal of cell biology.

[12]  S. Jackson,et al.  Cleavage and Inactivation of ATM during Apoptosis , 1999, Molecular and Cellular Biology.

[13]  J C Reed,et al.  Characterization of Caspase Processing and Activation in HL-60 Cell Cytosol Under Cell-free Conditions , 1999, The Journal of Biological Chemistry.

[14]  R. Newman,et al.  Oligomeric ring structure of the Bloom's syndrome helicase , 1999, Current Biology.

[15]  Leah E. Mechanic,et al.  Escherichia coli DNA Helicase II Is Active as a Monomer* , 1999, The Journal of Biological Chemistry.

[16]  Zhi-Min Yuan,et al.  Role for Caspase-Mediated Cleavage of Rad51 in Induction of Apoptosis by DNA Damage , 1999, Molecular and Cellular Biology.

[17]  G. Salvesen,et al.  Caspases: preparation and characterization. , 1999, Methods.

[18]  J. Noonan,et al.  The DNA helicase activity of BLM is necessary for the correction of the genomic instability of bloom syndrome cells. , 1999, Molecular biology of the cell.

[19]  G. Krockmalnic,et al.  The nuclear matrix prepared by amine modification. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[20]  A. Shimamoto,et al.  Cloning of two new human helicase genes of the RecQ family: biological significance of multiple species in higher eukaryotes. , 1998, Genomics.

[21]  C. Stroh,et al.  Death by a thousand cuts: an ever increasing list of caspase substrates , 1998, Cell Death and Differentiation.

[22]  I. Saira Mian,et al.  The premature ageing syndrome protein, WRN, is a 3′→5′ exonuclease , 1998, Nature Genetics.

[23]  J. Flygare,et al.  Proteolytic cleavage of HsRad51 during apoptosis , 1998, FEBS letters.

[24]  M MacFarlane,et al.  Different Subcellular Distribution of Caspase-3 and Caspase-7 following Fas-induced Apoptosis in Mouse Liver* , 1998, The Journal of Biological Chemistry.

[25]  M. Jezewska,et al.  Does Single-stranded DNA Pass through the Inner Channel of the Protein Hexamer in the Complex with the Escherichia coli DnaB Helicase? , 1998, The Journal of Biological Chemistry.

[26]  Alan G. Porter,et al.  Caspase-3 Is Required for DNA Fragmentation and Morphological Changes Associated with Apoptosis* , 1998, The Journal of Biological Chemistry.

[27]  F. Harmon,et al.  RecQ helicase, in concert with RecA and SSB proteins, initiates and disrupts DNA recombination. , 1998, Genes & development.

[28]  S. Nagata,et al.  A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD , 1998, Nature.

[29]  I. Hickson,et al.  The Bloom’s Syndrome Gene Product Is a 3′-5′ DNA Helicase* , 1997, The Journal of Biological Chemistry.

[30]  W. Earnshaw,et al.  Caspases and caspase inhibitors. , 1997, Trends in biochemical sciences.

[31]  G M Cohen,et al.  Caspases: the executioners of apoptosis. , 1997, The Biochemical journal.

[32]  N. Thornberry,et al.  Caspases: killer proteases. , 1997, Trends in biochemical sciences.

[33]  A. Shimamoto,et al.  DNA helicase activity in Werner's syndrome gene product synthesized in a baculovirus system. , 1997, Nucleic acids research.

[34]  N. Thornberry,et al.  A Combinatorial Approach Defines Specificities of Members of the Caspase Family and Granzyme B , 1997, The Journal of Biological Chemistry.

[35]  A. Porter,et al.  Death substrates come alive , 1997, BioEssays : news and reviews in molecular, cellular and developmental biology.

[36]  Yuri Lazebnik,et al.  Multiple species of CPP32 and Mch2 are the major active caspases present in apoptotic cells , 1997, The EMBO journal.

[37]  P. Nicotera,et al.  Intracellular Adenosine Triphosphate (ATP) Concentration: A Switch in the Decision Between Apoptosis and Necrosis , 1997, The Journal of experimental medicine.

[38]  K. Hanada,et al.  RecQ DNA helicase is a suppressor of illegitimate recombination in Escherichia coli. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[39]  P. Krammer,et al.  The CD95 system and the death of a lymphocyte. , 1997, Seminars in immunology.

[40]  Junying Yuan,et al.  Specific Cleavage of α-Fodrin during Fas- and Tumor Necrosis Factor-induced Apoptosis Is Mediated by an Interleukin-1β-converting Enzyme/Ced-3 Protease Distinct from the Poly(ADP-ribose) Polymerase Protease* , 1996, The Journal of Biological Chemistry.

[41]  Zhiyong Han,et al.  DNA-dependent Protein Kinase Is a Target for a CPP32-like Apoptotic Protease* , 1996, The Journal of Biological Chemistry.

[42]  D. Chan,et al.  DNA‐dependent protein kinase catalytic subunit: a target for an ICE‐like protease in apoptosis. , 1996, The EMBO journal.

[43]  David Wallach,et al.  Involvement of MACH, a Novel MORT1/FADD-Interacting Protease, in Fas/APO-1- and TNF Receptor–Induced Cell Death , 1996, Cell.

[44]  J. R. Smith,et al.  Inhibition of E2F activity by the cyclin-dependent protein kinase inhibitor p21 in cells expressing or lacking a functional retinoblastoma protein , 1996, Molecular and cellular biology.

[45]  N. Thornberry,et al.  Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death , 1996, The Journal of experimental medicine.

[46]  G. Schellenberg,et al.  Positional Cloning of the Werner's Syndrome Gene , 1996, Science.

[47]  D. Nicholson.  ICE/CED3-like Proteases as Therapeutic Targets for the Control of Inappropriate Apoptosis , 1996, Nature Biotechnology.

[48]  Ian D Hickson,et al.  Genome stability: Failure to unwind causes cancer , 1996, Current Biology.

[49]  N. Ellis,et al.  The Bloom's syndrome gene product is homologous to RecQ helicases , 1995, Cell.

[50]  C. Thompson,et al.  Apoptosis in the pathogenesis and treatment of disease , 1995, Science.

[51]  P. Blackshear,et al.  Cloning and characterization of RECQL, a potential human homologue of the Escherichia coli DNA helicase RecQ. , 1994, The Journal of biological chemistry.

[52]  T. Eki,et al.  Molecular cloning of cDNA encoding human DNA helicase Q1 which has homology to Escherichia coli Rec Q helicase and localization of the gene at chromosome 12p12. , 1994, Nucleic acids research.

[53]  Y. Lazebnik,et al.  Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE , 1994, Nature.

[54]  N. Werthamer.  Helping Physicists Market Themselves , 1993 .

[55]  M. Hingorani,et al.  Interactions of bacteriophage T7 DNA primase/helicase protein with single-stranded and double-stranded DNAs. , 1993, Biochemistry.

[56]  J. German.  Bloom Syndrome: A Mendelian Prototype of Somatic Mutational Disease , 1993, Medicine.

[57]  N. Davidson,et al.  Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early marker of chemotherapy-induced apoptosis. , 1993, Cancer research.

[58]  J. Schweizer,et al.  Simian virus 40 T-antigen DNA helicase is a hexamer which forms a binary complex during bidirectional unwinding from the viral origin of DNA replication , 1992, Journal of virology.

[59]  D. Compton,et al.  Identification of novel centromere/kinetochore-associated proteins using monoclonal antibodies generated against human mitotic chromosome scaffolds , 1991, The Journal of cell biology.

[60]  S. Adam,et al.  Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors , 1990, The Journal of cell biology.

[61]  D. Eisenberg.  Proteins. Structures and molecular properties, T.E. Creighton. W. H. Freeman and Company, New York (1984), 515, $36.95 , 1985 .

[62]  D. Wessel,et al.  A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. , 1984, Analytical biochemistry.

[63]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.

[64]  M. Lutter,et al.  Biochemical pathways of caspase activation during apoptosis. , 1999, Annual review of cell and developmental biology.

[65]  S H Kaufmann,et al.  Mammalian caspases: structure, activation, substrates, and functions during apoptosis. , 1999, Annual review of biochemistry.

[66]  K. Bjornson,et al.  Mechanisms of helicase-catalyzed DNA unwinding. , 1996, Annual review of biochemistry.

[67]  V. Fadok,et al.  Apoptosis and programmed cell death in immunity. , 1992, Annual review of immunology.

[68]  A. Wyllie,et al.  Apoptosis: mechanisms and roles in pathology. , 1991, International review of experimental pathology.

[69]  H. Horvitz,et al.  Mechanisms and functions of cell death. , 1991, Annual review of cell biology.