Possible link between the DNA damage checkpoint and chromatin remodelling factors

Beth Baber-Furnari

Appointment Period: 1999-2000 / Grant Year: [15]

The development of cancer is intimately linked to the failure to maintain genomic integrity. Completion of DNA synthesis and repair of DNA damage prior to chromosomal segregation is absolutely crucial to ensure genomic integrity and cell survival. Checkpoints are the safeguards that prevent cell cycle progression until DNA replication and repair are completed. The components of the checkpoint machinery are highly conserved from genetically tractable organisms such as Schizosaccharomyces pombe (S. pombe) to humans. Rad3, a kinase in S. pombe central to the checkpoint response, and its mammalian homologs, ATM and ATR, have a large N-terminal domain of over 2000 amino acids. Unfortunately, this domain contains very little information in terms of sequence motifs. The only potential 'functional' motifs include a leucine zipper, a nuclear localization sequence, a PCNA-like binding motif, and a single tetratricopeptide repeat. Additionally, ATM family members share a conserved region of approximately 600 amino acids. To determine if this region is required to elicit a checkpoint response, a series of N- and C-terminal truncations within this region in Rad3 were fused to GST under the control of an inducible promoter. Over expression of the fusion proteins in wild-type S. pombe did not affect cell growth, however, the DNA damage and replication checkpoints were severely compromised upon overproduction of some of these fusions. The minimal region required for the interference with the checkpoint response contains a LXCXE motif. This sequence in ATM was recently shown to be required for its association with a histone deacetylase, HDAC1. A cysteine to phenylalanine mutation prevents the dominant negative phenotype observed above in S. pombe and also, the association of ATM with HDAC1. These results highly suggest an 'in vivo' role for histone deacetylases (HDACs) in the checkpoint response to abnormal DNA structures such as double-strand breaks and stalled replication forks. Therefore, it is important to further investigate the physiological role of this interaction between chromatin remodeling factors such as HDACs and the checkpoint proteins ATM, ATR, and Rad3. Currently, I am in the process of determining an 'in vivo' role for histone deacetylases in both fission yeast and mammalian DNA structure checkpoints.

The mechanism by which these checkpoint kinases recognize abnormal DNA structures such as double-strand breaks and stalled replication forks remains unknown. ATM has been shown to bind to double-stranded DNA in vitro and its association is enhanced in the presence of breaks in the DNA. It is unknown whether ATM or ATR is actually recruited to sites of abnormal DNA structures. I am currently establishing a chromatin immunoprecipitation assay in S. pombe to determine if Rad3 associates with a single double-stranded break in vivo, and what repair and checkpoint factors are required for the association.

SPECIAL NOTE: The UCSD Rady School of Management has named Beth A. Baber as the recipient of the 2007 DLA Piper-Athena FlexMBA Scholarship. Dr. Baber is in the process of establishing The Nicholas Conor Institute for Pediatric Cancer Research, named after her son who was diagnosed with neuroblastoma in 2005. For the complete article click: here.

PUBLICATIONS (resulting from this training)

Rhind N, Baber-Furnari BA, Lopez-Girona A, Boddy MN, Brondello JM, Moser B, Shanahan P, Blasina A, McGowan C, Russell P. (2000) DNA Damage Checkpoint Control of Mitosis in Fission Yeast. Cold Spring Harbor Symp. Quant. Biol. 65:353-9.

Baber-Furnari BA, Rhind N, Boddy MN, Shanahan P, Lopez-Girona A, Russell P. (2000) Regulation of mitotic inhibitor Mik1 helps to enforce the DNA damage checkpoint. Mol Biol Cell. 11:1-11.

Moser BA, Brondello JM, Baber-Furnari B, Russell P. (2000) Mechanism of caffeine induced checkpoint override in fission yeast. Mol Cell Biol. 20:4288-4294.

Lopez-Girona A, Tanaka K, Chen XB, Baber BA, McGowan CH, Russell P. (2001) Serine-345 is required for Rad3-dependent phosphorylation and function of checkpoint kinase Chk1 in fission yeast. Proc Natl Acad Sci USA. 98:11289-11294.