ABSTRACT
DNA polymerase beta (Pol ß) is a key enzyme for the protection against oxidative DNA lesions via its role in base excision repair (BER). Approximately 1/3 of tumors studied to date express Pol ß variant proteins, and several tumors overexpress Pol ß. Pol ß possesses DNA polymerase and dRP lyase activities, both of which are known to be important for efficient BER. The dRP lyase activity resides within the 8kDa amino terminal domain of Pol ß, is responsible for removal of the 5' phosphate group (5'-dRP). The DNA polymerase subsequently fills the gaps. Previously, we demonstrated that the human gastric cancer-associated variant of Pol ß (Leu22Pro (L22P)) lacks dRP lyase function in vitro. Here, we report that L22P-expressing cells harbor significantly increased replication associated DNA double strand breaks (DSBs) and defective maintenance of the nascent DNA strand (NDS) during replication stress. Moreover, L22P-expressing cells are sensitive to PARP1 inhibitors, which suggests trapped PARP1 binds to the 5'-dRP group and blocks replications forks, resulting in fork collapse and DSBs. Our data suggest that the normal function of the dRP lyase is critical to maintain replication fork integrity and prevent replication fork collapse to DSBs and cellular transformation.
Subject(s)
DNA Breaks, Double-Stranded , DNA Polymerase beta/genetics , DNA Repair/genetics , DNA Replication/genetics , Stomach Neoplasms/genetics , Cell Line, Tumor , DNA, Single-Stranded/genetics , Humans , Phosphorus-Oxygen Lyases/genetics , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerases/metabolism , Protein Isoforms/genetics , Rad51 Recombinase/biosynthesisABSTRACT
BACKGROUND: Fetal alcohol spectrum disorders (FASD) are a highly variable set of phenotypes caused by fetal alcohol exposure. Numerous factors influence FASD phenotypes, including genetics. The zebrafish is a powerful vertebrate model system with which to identify these genetic factors. Many zebrafish mutants are housed at the Zebrafish International Resource Center (ZIRC). These mutants are readily accessible and an excellent source to screen for ethanol (EtOH)-sensitive developmental structural mutants. METHODS: We screened mutants obtained from ZIRC for sensitivity to EtOH teratogenesis. Embryos were treated with 1% EtOH (41 mM tissue levels) from 6 hours postfertilization onward. Levels of apoptosis were evaluated at 24 hours postfertilization. At 4 days postfertilization, the craniofacial skeleton, peripheral axon projections, and sensory neurons of neuromasts were examined. Fish were genotyped to determine whether there were phenotype/genotype correlations. RESULTS: Five of 20 loci interacted with EtOH. Notable among these was that vangl2, involved in convergent extension movements of the embryonic axis, interacted strongly with EtOH. Untreated vangl2 mutants had normal craniofacial morphology, while severe midfacial defects including synophthalmia and narrowing of the palatal skeleton were found in all EtOH-treated mutants and a low percentage of heterozygotes. The cell cycle gene, plk1, also interacted strongly with EtOH. Untreated mutants have slightly elevated levels of apoptosis and loss of ventral craniofacial elements. Exposure to EtOH results in extensive apoptosis along with loss of neural tissue and the entire craniofacial skeleton. Phenotypes of hinfp, mars, and foxi1 mutants were also exacerbated by EtOH. CONCLUSIONS: Our results provide insight into the gene-EtOH interactions that may underlie EtOH teratogenesis. They support previous findings that EtOH disrupts elongation of the embryonic axis. Importantly, these results show that the zebrafish is an efficient model with which to test for gene-EtOH interactions. Understanding these interactions will be crucial to understanding of the FASD variation.
