St Vincent's Institute

Molecular Genetics - Research Units - Molecular Genetics - Research themes

Function and regulation of CHK2-like checkpoint kinases

CHK2-like kinases, mutations in which can lead to elevated cancer risk in humans, are characterized by N-terminal SQ/TQ cluster and FHA domains that function as phosphorylation-dependent protein-protein interaction modules. The yeast Rad53 kinase is a unique member of this family in that it contains a second C-terminal FHA domain.ﾠ We have found that these two FHA domains have largely overlapping functions in the regulation of Rad53 activation by DNA damage during most cell cycle phases.ﾠ However, the N-terminal FHA1 domain has important non-redundant functions in Rad53 activation during metaphase, and also in linking activated Rad53 to downstream effectors to regulate DNA replication during S phase.ﾠ In addition, by isolating binding partners for the yeast Rad53 FHA1 domain and the human CHK2 FHA domain, we have identified yeast Mdt1 and human ASCIZ as novel related DNA damage response proteins.ﾠ In collaboration with Ming-Daw Tsai's lab we recently found that the Dun1 kinase FHA domain interacts with an SQ/TQ cluster domain of Rad53 in an unprecedented di-phospho-threonine-specific manner that regulates Dun1 activation by Rad53. We are continuing to study the regulation and function of Chk2-like kinases in response to DNA damaging as well as during normal unperturbed cell cycles.

Selected Publications
Pike BL, Yongkiettrakul S, Tsai M-D & Heierhorst J (2003). Diverse but overlapping functions of the two forkhead-associated (FHA) domains in Rad53 checkpoint kinase activation.ﾠ J. Biol. Chem. 278, 30421-30424.

Tam ATY, Pike BL & Heierhorst J (2008).ﾠ Location-specific functions of the two FHA domains in Rad53 checkpoint kinase signaling.ﾠ Biochemistry 47, 3912-3916.

Lee H, Yuan C, Hammet A, Mahajan A, Wu MR, Chen ESW, Su MI, Heierhorst J & Tsai MD (2008). Diphosphothreonine-specific interaction between an SQ/TQ cluster and an FHA domain in the Rad53-Dun1 kinase cascade. Mol. Cell 30, 767-778.

DNA damage responses in budding yeast

Key elements of the DNA damage response are highly conserved throughout evolution, which allows us to employ yeast as a simple model organism to study fundamental mechanisms relevant for human cancer biology. Our main interests in this area are: (a) analysis of the regulation of the yeast CHK2-like kinase Rad53 and how this contributes to cell cycle delays in order to allow time for DNA repair; and (b) interplay between DNA damage response and telomere maintenance pathways.ﾠ In addition, we have identified a link between another yeast CHK2-like kinase, Dun1, to proteins involved in regulating mRNA stability and translation into protein.ﾠ We have found that Dun1 interacts with the Pan2/3 and Ccr4-Caf1 mRNA deadenylases whose catalytic activities are required for normal DNA damage tolerance.

Selected Publications
Hammet A, Pike BL & Heierhorst J (2002). Posttranscriptional regulation of the RAD5 DNA repair gene by the Dun1 kinase and the Pan2/Pan3 poly(A)-nuclease complex contributes to survival of replication blocks. J. Biol. Chem. 277, 22469-22474. [reviewed in F1000]

Pike BL, Yongkiettrakul S, Tsai M-D & Heierhorst J (2004). Mdt1, a novel Rad53 FHA1 domain-interacting protein, modulates DNA damage tolerance and G2/M cell cycle progression in Saccharomyces cerevisiae. Mol. Cell. Biol. 24, 2779-2788.

Traven A, Hammet A, Tenis N, Denis CL & Heierhorst J (2005). Ccr4-Not complex mRNA deadenylase activity contributes to DNA damage responses in Saccharomyces cerevisiae. Genetics 169, 65-75.

Pike BL & Heierhorst J (2007).ﾠ Mdt1 facilitates efficient repair of blocked DNA double-strand breaks and recombinational maintenance of telomeres.ﾠ Mol. Cell. Biol. 27, 6532-6545.

Roles of ASCIZ in DNA repair

ASCIZ (ATM substrate Chk2-interacting Zn²⁺-finger protein) is a new SQ/TQ cluster domain protein identified in our laboratory, and structurally related to yeast Mdt1. ASCIZ is normally diffusely distributed throughout the nucleus in human cells, but redistributes to characteristic DNA damage-induced nuclear foci in response to methylating and oxidative base damage. Human, chicken or mouse cells that lack ASCIZ are 5-10-fold more sensitive to methylating and oxidative DNA damage, indicating that these foci are important for DNA repair. Ongoing work in the laboratory is directed at deciphering ASCIZ foci components and mechanisms how they contribute to DNA repair in a highly lesion-specific manner.

Selected Publications
McNees CJ, Conlan LA, Tenis N & Heierhorst J (2005).ﾠ ASCIZ regulates lesion-specific RAD51 focus formation and apoptosis after methylating DNA damage.ﾠ EMBO J. 24, 2447-2457.ﾠ

Oka H, Sakai W, Sonoda E, Nakamura J, Asagoshi K, Wilson SH, Kobayashi M, Yamamoto K, Heierhorst J, Takeda S & Taniguchi Y (2008). DNA damage response protein ASCIZ links base excision repair with immunoglobulin gene conversion. Biochem. Biophys. Res. Comm. 371, 225-229.

Heierhorst J (2008). Mdt1/ASCIZ -a new DNA damage response protein family. Cell Cycle 7, 2654-2660.

Jurado S, Smyth I, Van Denderen B, Tenis N, Hammet A, Hewiitt K, Ng JL, McNees CJ, Kozlov SV, Oka H, Kobayashi M, Conlan LA, Cole TC, Yamamoto K, Taniguchi Y, Takeda S, Lavin MF & Heierhorst J (2010). Dual functions of ASCIZ in the DNA base damage response and pulmonary organogenesis. PLoS Genetics 6, e1001170. [http://www.plosgenetics.org/doi/pgen.1001170]

Roles of ASCIZ in organ development

We recently found that mice lacking the gene for the DNA repair protein ASCIZ (ATM substrate Chk2-interacting Zn²⁺-finger protein) die late during embryonic development with a range of organ defects. Most strikingly, ASCIZ-deficient embryos completely fail to develop lungs and also have severe tracheal defects. Our data indicate that this unexpected function of ASCIZ is unrelated to its role in the DNA damage response. Instead, ASCIZ seems to have a separate function as a crucial Zinc-finger transcription factor during organ development, including as a designated transcription factor for the expression of the dynein light chain DYNLL1. We found that ASCIZ binds to the Dynll1 promoter in primary cells and regulates its expression in a Zn²⁺-finger-dependent manner. In turn, DYNLL1 protein can bind to at least 10 sites in the ASCIZ transcription activation domain and downregulates its activity in a concentration-dependent manner. Thus, the dual ability of ASCIZ to activate Dynll1 gene expression and to sense free DYNLL1 protein levels enables a simple dynamic feedback loop to adjust DYNLL1 levels to cellular needs. Ongoing projects are directed at determining to what extent reduced DYNLL1 levels are responsible for developmental defects of ASCIZ KO mice.

Selected Publications
Jurado S, Smyth I, Van Denderen B, Tenis N, Hammet A, Hewiitt K, Ng JL, McNees CJ, Kozlov SV, Oka H, Kobayashi M, Conlan LA, Cole TC, Yamamoto K, Taniguchi Y, Takeda S, Lavin MF & Heierhorst J (2010). Dual functions of ASCIZ in the DNA base damage response and pulmonary organogenesis. PLoS Genetics 6, e1001170. [http://www.plosgenetics.org/doi/pgen.1001170]