title: Coordination of the Ser2056 and Thr2609 Clusters of DNA-PKcs in Regulating Gamma Rays and Extremely Low Fluencies of Alpha-Particle Irradiation to G0/G1Phase Cells.
abstract: 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. In this study, we further examine phenotypic effects on cells bearing various combinations of mutations within either or both regions. Effects studied included cell killing as well as chromosomal aberration induction after 0.5-8 Gy gamma-ray irradiation delivered to synchronized cells during the G0/G1phase of the cell cycle. Blocking phosphorylation within the T2609 cluster was most critical regarding sensitization and depended on the number of available phosphorylation sites. It was also especially interesting that only one substitution of alanine in each of the two clusters separately abolished the restoration of wild-type sensitivity by DNA-PKcs. Similar patterns were seen for induction of chromosomal aberrations, reflecting their connection to cell killing. To study possible change in coordination between HRR and NHEJ directed repair in these DNA-PKcs mutant cell lines, we compared the induction of sister chromatid exchanges (SCEs) by very low fluencies of alpha particles with mutant cells defective in the HRR pathway that is required for induction of SCEs. Levels of true SCEs induced by very low fluence of alpha-particle irradiation normally seen in wild-type cells were only slightly decreased in the S2056 cluster mutants, but were completely abolished in the T2609 cluster mutants and were indistinguishable from levels seen in HRR deficient cells. Again, a single substitution in the S2056 together with a single substitution in the T2609 cluster abolished SCE formation and thus also effectively interferes with HRR.
authors.affiliation: a   Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523.
authors.forename: Hatsumi
authors.initials: H
authors.lastname: Nagasawa
authors.affiliation: b   Department of Radiation Oncology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390.
authors.forename: Yu-Fen
authors.initials: YF
authors.lastname: Lin
authors.affiliation: a   Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523.
authors.forename: Takamitsu A
authors.initials: TA
authors.lastname: Kato
authors.affiliation: a   Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523.
authors.forename: John R
authors.initials: JR
authors.lastname: Brogan
authors.affiliation: b   Department of Radiation Oncology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390.
authors.forename: Hung-Ying
authors.initials: HY
authors.lastname: Shih
authors.affiliation: c   Tohoku Medical and Pharmaceutical University, Sendai, Japan, 981-8558; and.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kurimasa
authors.affiliation: a   Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523.
authors.forename: Joel S
authors.initials: JS
authors.lastname: Bedford
authors.affiliation: b   Department of Radiation Oncology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390.
authors.forename: Benjamin P C
authors.initials: BP
authors.lastname: Chen
authors.affiliation: d   Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, Massachusetts 02115.
authors.forename: John B
authors.initials: JB
authors.lastname: Little
mesh_terms: D000512:Alpha Particles / Q000009:adverse effects*; D000818:Animals; D016466:CHO Cells; D002869:Chromosome Aberrations / Q000528:radiation effects; D006224:Cricetinae; D003412:Cricetulus; D051747:DNA-Activated Protein Kinase / Q000737:chemistry / Q000378:metabolism*; D016193:G1 Phase / Q000528:radiation effects*; D005720:Gamma Rays / Q000009:adverse effects*; D016192:Resting Phase, Cell Cycle / Q000528:radiation effects*; D012694:Serine / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*
chemical_list: D013912:Threonine; D012694:Serine; D051747:DNA-Activated Protein Kinase