Abstract
Ku70-Ku80 complex is the regulatory subunit of DNA-dependent protein kinase (DNA-PK) and plays an essential role in double-strand break repair following ionizing radiation (IR). It preferentially interacts with chromosomal breaks and protects DNA ends from nuclease attack. Here we show evidence that cells defective in Ku80 exhibit a significantly slow S phase progression following DNA damage. IR-induced retardation in S phase progression in Ku80-/- cells was not due to the lack of DNA-PK kinase activity because both wild-type cells and DNA-PKcs-deficient cells showed no such symptom. Instead, proliferating cell nuclear antigen (PCNA) dissociated from chromosomes following IR in Ku80-deficient cells but not in wild-type or DNA-PKcs-deficient cells. Treatment of HeLa cells with IR induced colocalization of the Ku complex with PCNA on chromosomes. Together, these results suggest that binding of the Ku complex at chromosomal breaks may be necessary to maintain the sliding clamps (PCNA) on chromatin, which would allow cells to resume DNA replication without a major delay following IR.
Highlights
Upon DNA damage, eukaryotic cells invoke several mechanisms that control DNA metabolism as well as cell cycle progression [1,2,3]
Ku80-deficient Cells Exhibit a Prolonged Retardation in DNA Replication Following IR—DNA-PK and ATM are the members of PI 3-kinase family involved in damage-induced cell relative amount of [3H]thymidine incorporation in irradiated cells compared with non-irradiated cells
A prolonged delay of Ku80Ϫ/Ϫ cells in S phase progression following IR was different from the ATM-mediated intra-S phase checkpoint pathway (Fig. 2B) that exhibits a transient replication arrest [10, 11]. These results suggest the following: 1) IR-induced inhibition of S phase progression in Ku80Ϫ/Ϫ cells is not related to G1/S phase or the intra-S phase checkpoint pathway; 2) the Ku complex, unlike DNA-PKcs or ATM, may have a unique role in DNA replication following DNA damage
Summary
Upon DNA damage, eukaryotic cells invoke several mechanisms that control DNA metabolism as well as cell cycle progression [1,2,3]. DNA-dependent protein kinase (DNA-PK) is a member of the PI 3-kinase family that is regulated by various stresses and shares amino acid sequence homology in its C-terminal kinase domain with other family members (ATM, ATM-related, and p110 PI 3-kinase) [20, 21] It is a nuclear serine/threonine protein kinase that is involved in the damage checkpoint pathway but is an essential factor for double-strand break (dsb) repair ( known as non-homologous end-joining (NHEJ) repair). It is a three-protein complex consisting of a 460-kDa catalytic subunit (DNA-PKcs) and two regulatory subunits (Ku70/Ku80 heterodimer). The abbreviations used are: PI, phosphatidylinositol; DNA-PK, DNAdependent protein kinase; dsb, double-strand break; IR, ionizing radiation; WT, wild type; PCNA, proliferating cell nuclear antigen; BrdUrd, bromodeoxyuridine; Gy, gray; DTT, dithiothreitol; PBS, phosphatebuffered saline; FITC, fluorescein isothiocyanate; ATM, ataxia telangiectasia mutated; RDS, radio-resistant DNA synthesis
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