Biological response to ionizing radiation in mouse embryo fibroblasts witha targeted disruption of the DNA polymerase beta gene

Base excision repair (BER) is carried out by two distinct pathways in mamma lian cells, one dependent on DNA polymerase beta (Polb) and the other on pr oliferating cell nuclear antigen (Pcna), We studied whether the Polb-depend ent pathway plays an important role in BER in vivo after exposure to ionizi ng radiation. For this purpose, we used mouse embryo filbroblasts derived f rom wild-type and Polb gene knockout littermates, Both cell lines had essen tially the same clonogenic cell survival and low levels of apoptosis as det ermined by a colony formation assay and by a change in mitochondrial membra ne potential, respectively, No significant cleavage of protein kinase C del ta (Pkcd) in vivo, which is a substrate for caspase 3, was detected, and in tact Pkcd was retained in both cell lines for at least 72 h after irradiati on. Similar significant increases in caspase 3-like activities as measured by Asp-Glu-Val-Asp (DEVD) cleaving activity ill vitro were observed in both cell lines after Irradiation. Radiation induced cell cycle arrest in the f orm of a G(2)-phase block, and G(2)/M-phase fractions reached a peak approx imately 10 h after irradiation and decreased thereafter with a similar time course in both cell lines. Similar levels of chromatin-bound Pcna were obs erved immediately after irradiation in non-S.phase cells of both cell lines and disappeared by 4 h after irradiation, We conclude that the deficiency in Polb does not have a significant influence on the radiation responses of these cells. Together with evidence accumulated in vitro, these results st rongly support the idea that the Pcna-dependent pathway predominantly acts in BER of radiation-induced DNA damage in vivo. (C) 2000 by Rndintion Resea rch Society.