Endogenous gamma-H2AX-ATM-Chk2 checkpoint activation in Bloom's syndrome helicase deficient cells is related to DNA replication arrested forks.
The Bloom syndrome helicase (BLM) is critical for genomic stability. A defect in BLM activity results in the cancer-predisposing Bloom syndrome (BS). Here, we report that BLM-deficient cell lines and primary fibroblasts display an endogenously activated DNA double-strand break checkpoint response with prominent levels of phosphorylated histone H2AX (gamma-H2AX), Chk2 (p(T68)Chk2), and ATM (p(S1981)ATM) colocalizing in nuclear foci. Interestingly, the mitotic fraction of gamma-H2AX foci did not seem to be higher in BLM-deficient cells, indicating that these lesions form transiently during interphase. Pulse labeling with iododeoxyuridine and immunofluorescence microscopy showed the colocalization of gamma-H2AX, ATM, and Chk2 together with replication foci. Those foci costained for Rad51, indicating homologous recombination at these replication sites. We therefore analyzed replication in BS cells using a single molecule approach on combed DNA fibers. In addition to a higher frequency of replication fork barriers, BS cells displayed a reduced average fork velocity and global reduction of interorigin distances indicative of an elevated frequency of origin firing. Because BS is one of the most penetrant cancer-predisposing hereditary diseases, it is likely that the lack of BLM engages the cells in a situation similar to precancerous tissues with replication stress. To our knowledge, this is the first report of high ATM-Chk2 kinase activation and its linkage to replication defects in a BS model.
Rao
V Ashutosh
VA
Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, U.S. Department of Health and Human Services, Bethesda, Maryland, USA.
Conti
Chiara
C
Guirouilh-Barbat
Josee
J
Nakamura
Asako
A
Miao
Ze-Hong
ZH
Davies
Sally L
SL
Saccá
Barbara
B
Hickson
Ian D
ID
Bensimon
Aaron
A
Pommier
Yves
Y
D000251:Adenosine Triphosphatases / Q000172:deficiency*; D064007:Ataxia Telangiectasia Mutated Proteins; D001816:Bloom Syndrome / Q000201:enzymology / Q000235:genetics / Q000473:pathology*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002461:Cell Line, Transformed; D064447:Checkpoint Kinase 2; D004265:DNA Helicases / Q000172:deficiency*; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008677:Metaphase; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport; D051135:Rad51 Recombinase / Q000378:metabolism; D053484:RecQ Helicases; D011995:Recombination, Genetic / Q000235:genetics; D018741:Replication Origin; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D010750:Phosphoproteins; D025521:Tumor Suppressor Proteins; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D000251:Adenosine Triphosphatases; C097151:Bloom syndrome protein; D004265:DNA Helicases; D053484:RecQ Helicases; D004264:DNA Topoisomerases, Type I