Reactive oxygen species and electrophiles arising from endogenous and environmental sources can induce single and double strand breaks in DNA. These DNA lesions activate a multifaceted DNA damage response (DDR) to promote DNA repair by respective single and double strand break repair pathways (SSBR and DSBR). To ensure fidelity during DNA replication, the repair of single and double strand breaks is critical for genome maintenance and chromosome stability. If left unrepaired, these DNA lesions can result in cell death or mutations. If these mutations disrupt the function of key regulatory genes involved in cell growth and homeostasis, premature aging or cancer can occur. Indeed, defects in components of the DDR, such as ATM, BRCA1 and p53 are known to cause a predisposition to cancer development as well as hypersensitivity to ultraviolet and ionizing radiation [1-3]. Therefore, a better understanding of genome maintenance and DNA repair processes are important areas of research. The DDR is a broad cellular response involving a network of proteins that are highly conserved between yeast and mammals . Recently, we discovered a new link between protein translation and the DDR by establishing that yeast Trm9-catalyzed tRNA modifications up-regulate the levels of several DNA damage response proteins through enhanced translation . Our interest in mammalian Abh8 stems from comparative sequence analysis that indicated it is a homolog of yeast Trm9 and our data demonstrating that Abh8 is a likely translational component of DDR. These results include data demonstrating: i) Abh8 deletion in human and mouse cells increases cellular sensitivity to multiple DNA damaging agents, ii) Abh8-/- MEFs show decreased proliferation and colony forming potential compared to wildtype MEFs, iii) human cells depleted of ATM show decreased Abh8 induction after DNA damage, and iv) p53 levels are elevated in Abh8-/- MEFs compared to wild-type MEFs. The following aims intend to help elucidate the role of mammalian Abh8 in genome maintenance. Specifically, we hypothesize that mammalian Abh8 optimizes the DDR signaling network by regulating the translation of proteins involved in single or double strand break repair, thereby ensuring proper genome maintenance. To test this hypothesis we propose the following two aims:
AIM 1 : Determine if there are increased levels of DNA damage in Abh8-/- MEFs caused by corrupted DNA repair Since Abh8-/- MEFs have an impaired growth and colony forming phenotype, the levels of DNA damage will be assessed in Abh8-/- MEFs relative to their wildtype counterparts. We predict that the slow growth and diminished colony forming phenotype of Abh8-/- MEFs arises from an inability to tolerate endogenous DNA damage. To assess levels of DNA damage, we will use Fluorescence In Situ Hybridization (FISH) staining with H2AX to detect DNA strand breaks and use COMET assays as a quantitative measurement of DNA strand breaks. As a further perturbation that may enhance a defective basal DNA repair phenotype, similar experiments will be performed after exposure of MEFs to ionizing radiation. We expect that the deletion of Abh8-/- in our mouse model will lead to defects in DNA repair and that this will eventually lead to widespread genomic instability and cancer predisposition. This contention is supported by the observation that large single colonies of Abh8-/- MEFs emerge after long-term growth in culture. I propose to isolate these colonies and assay them for chromosomal abnormalities using spectral karyotyping. Additionally, to investigate a potential cancer predisposition phenotype, Abh8-/- mice will be monitored for spontaneous tumor development for up to 1 year. To complement any cancer phenotype we will use whole body radiation to initiate tumor development and investigate DNA repair capacity in radiosensitive tissues (i.e., thymic B-cells and intestinal crypt cells) by H2AX staining and using the COMET assay. Finally, we will measure the expression levels of DDR component transcripts and proteins in Abh8-/- and wild-type MEFs, both under basal growth conditions and in response to DNA damage. Affymetrix GeneChip and high throughput qPCR arrays (SABiosciences/Qiagen") will be used to determine the levels of DDR transcripts, while the status of key proteins involved in SSBR and DSBR repair will be determined by Western blot analysis (i.e., for SSBR, PARP, XRCC1 and polynucleotide kinase;for DSBR, RAD family proteins, XRCC2 and 3, BRCA1 and 2, Ku70 and 86, and DNAPK). Mammalian Abh8 is known to have a similar biochemical function as yeast Trm9;therefore, we expect that many markers of DNA damage will be altered at the protein rather than at the transcript level. The combined approaches of transcription-based arrays and Western blot analysis should elucidate whether this proves to be the case.
AIM 2 : Determine how ATM and p53 dependent signaling pathways regulate Abh8 Our preliminary data suggests that Abh8 is connected to both ATM and p53 signaling;therefore, we propose to fully characterize Abh8 transcript and protein levels within the ATM and p53 DDR network, in both human and mouse contexts. We predict that Abh8 expression levels will be altered in an ATM- or p53-dependent manner. Abh8 transcript and protein levels will be assessed by qPCR and Western blot analysis, respectively, in a panel of cells rendered ATM or p53 deficient by chemical or genetic approaches. Specifically, we will use MEFs treated with chemical inhibitors of ATM (Santa Cruz 202963) and p53 (i.e., Pifithrin 1);ATM-/- and p53-/- MEFs;human AT fibroblasts and Li Fraumeni lymphoblastoid cells. Based on predicative software analysis (ExPASy), there are a number of conserved phosphorylation and kinase recognition sites in human and mouse Abh8. Several of these kinases act in DDR signaling (i.e., CK1, CK2, DNAPK and ATM), suggesting that Abh8 is a likely candidate for post-translational modification during the DDR. Our preliminary immunoblots support phosphorylation of Abh8 after DNA damage. We will use further immunoblots and Q- TOF LC/MS to identify cell cycle and DDR specific post-translational (PT) modifications of Abh8. Using this approach, we expect to identify novel DNA damage-induced Abh8 post-translational modifications that can be mapped back to consensus kinase sites to identify DDR signaling pathways linked to Abh8. Overall, these findings will form the basis of future studies into Abh8 regulation during the DDR.
The main objectives of this proposal are to analyze the role of mammalian Abh8 in maintaining genome integrity and to determine if Abh8 contributes to a translational component of the DNA damage response (DDR). Specifically, I hypothesize that mammalian Abh8 optimizes the DDR signaling network by regulating the translation of proteins involved in single or double strand break repair, thereby ensuring proper genome maintenance. I propose to use molecular and systems based studies, as well as animal studies, to understand the role of Abh8 in preventing DNA damage-induced cellular problems and disease. The long term potential of the proposed research is the use of Abh8 activity as a marker for susceptibility to environmentally induced disease.
|Endres, Lauren; Dedon, Peter C; Begley, Thomas J (2015) Codon-biased translation can be regulated by wobble-base tRNA modification systems during cellular stress responses. RNA Biol 12:603-14|
|Begley, Ulrike; Sosa, Maria Soledad; Avivar-Valderas, Alvaro et al. (2013) A human tRNA methyltransferase 9-like protein prevents tumour growth by regulating LIN9 and HIF1-Î±. EMBO Mol Med 5:366-83|