Coordination of the Ser2056 and Thr2609 Clusters of DNA-PKcs in Regulating Gamma Rays and Extremely Low Fluencies of Alpha-Particle Irradiation to G/G Phase Cells.

The catalytic subunit of DNA dependent protein kinase (DNA-PKcs) and its kinase activity are critical for mediation of non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB) in mammalian cells after gamma-ray irradiation. Additionally, DNA-PKcs phosphorylations at the T2609 cluster and the S2056 cluster also affect DSB repair and cellular sensitivity to gamma radiation. Previously we reported that phosphorylations within these two regions affect not only NHEJ but also homologous recombination repair (HRR) dependent DSB repair.

Differential radiosensitivity phenotypes of DNA-PKcs mutations affecting NHEJ and HRR systems following irradiation with gamma-rays or very low fluences of alpha particles.

We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase.

Differential role of DNA-PKcs phosphorylations and kinase activity in radiosensitivity and chromosomal instability.

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is the key functional element in the DNA-PK complex that drives nonhomologous end joining (NHEJ), the predominant DNA double-strand break (DSB) repair mechanism operating to rejoin such breaks in mammalian cells after exposure to ionizing radiation. It has been reported that DNA-PKcs phosphorylation and kinase activity are critical determinants of radiosensitivity, based on responses reported after irradiation of asynchronously dividing populations of various mutant cell lines. In the present study, the relative radiosensitivity to cell killing as well as chromosomal instability of 13 DNA-PKcs site-directed mutant cell lines (defective at phosphorylation sites or kinase activity) were examined after exposure of synchronized G(1) cells to (137)Cs γ rays.

Some unsolved problems and unresolved issues in radiation cytogenetics: a review and new data on roles of homologous recombination and non-homologous end joining.

New data and historical evidence from our own and other laboratories are summarized and discussed bearing on several issues relating to mechanisms and processes involved in the formation of chromosomal aberrations following exposure to ionizing radiations. Specifically addressed are: (1) the lesions and processes affecting the appearance of chromatid-type and/or chromosome-type aberrations after radiation, (2) DNA double strand break rejoining processes and the restitution of breaks vs. the formation of exchanges, (3) the role of homologous recombinational repair in protecting cells from induction of chromatid-type aberrations after irradiation of late S/G2 cells, (4) the role of interphase chromatin structure and nuclear organization in aberration induction, (5) cellular responses for aberration induction in relation to their tissue context, and (6) approaches to the detection of aberrations previously known as "cryptic".