Oxidative damage (OxD) to DNA is a normal occurrence, the result of both endogenous and exogenous events. It is repaired by the base excision repair (BER) pathway, which is present in all cells. BER has now been well characterized in the test tube, in yeast, and in mammalian cell lines. Pathway entry is via a glycosylase, e.g., OGG1 or UDG, that recognizes the damage, e.g., 8-oxoG or the presence of uracil. AP endonuclease (AP endo) then generates the 3' hydroxyl so that the repair polymerase can insert the correct nucleotide, while a polymerase, usually DNA polymerase-beta (pol-beta) inserts the missing nucleotide and removes the phosphodeoxyribose. While there are redundancies for the glycosylases and mouse knock-out models show no or weak phenotypes, APendo1 provides 95% of the mammalian cell's ability to prepare an abasic site for repair, and the homozygous knock-out in mice is lethal. Knock-out for pol-beta results in death at parturition with abnormal neurological development. Nothing is known about BER in normal development. Since murine embryos deficient in BER die at two different stages, seven to ten days after knock-out of AP endo1 (gastrulation) or parturition, the investigators hypothesize that BER proteins are important at two distinct stages in development. They propose to examine BER during development in zebrafish, where there is easy access to multiple embryos at different stages of development, a genomic project that is well underway, and the ability to knock-down protein expression efficiently in the early stages of development using morpholino derivatives of RNA. In fact, the investigators have already cloned and expressed the gene for AP endo1 in zebrafish, and shown that there are two mRNAs with the same coding sequence but distinct 3'-UTRs.
Specific Aim 1 is to measure BER at different stages of zebrafish development, and Specific Aim 2 is to knock-down zfAPE1 followed by rescue to determine whether lethality is related to BER.
For Aim 1 the investigator will apply standard BER and abasic site assays to embryos and examine how BER changes during normal development.
For Aim 2 they will examine how knock-down of zfAPE1 and rescue with different constructs affects viability and apoptosis, number, and distribution of abasic sites, BER, and morphogenesis. These studies will help define the role of BER in embryogenesis and open a new field of inquiry. ? ? ?
| Fortier, Sean; Yang, Xiaojie; Wang, Yi et al. (2009) Base excision repair in early zebrafish development: evidence for DNA polymerase switching and standby AP endonuclease activity. Biochemistry 48:5396-404 |
| Wang, Yi; Shupenko, Craig C; Melo, Luisa F et al. (2006) DNA repair protein involved in heart and blood development. Mol Cell Biol 26:9083-93 |
