Induction of persistent double strand breaks following multiphoton irradiation of cycling and G1-arrested mammalian cells-replication-induced double strand breaks.
DNA double strand breaks (DSBs) are amongst the most deleterious lesions induced within the cell following exposure to ionizing radiation. Mammalian cells repair these breaks predominantly via the nonhomologous end joining pathway which is active throughout the cell cycle and is error prone. The alternative pathway for repair of DSBs is homologous recombination (HR) which is error free and active during S- and G2/M-phases of the cell cycle. We have utilized near-infrared laser radiation to induce DNA damage in individual mammalian cells through multiphoton excitation processes to investigate the dynamics of single cell DNA damage processing. We have used immunofluorescent imaging of gamma-H2AX (a marker for DSBs) in mammalian cells and investigated the colocalization of this protein with ATM, p53 binding protein 1 and RAD51, an integral protein of the HR DNA repair pathway. We have observed persistent DSBs at later times postlaser irradiation which are indicative of DSBs arising at replication, presumably from UV photoproducts or clustered damage containing single strand breaks. Cell cycle studies have shown that in G1 cells, a significant fraction of multiphoton laser-induced prompt DSBs persists for > 4 h in addition to those induced at replication.
DNA Damage Group, Radiation Oncology Biology, Old Road Campus Research Building, University of Oxford, Oxford, UK. jane.harper@rob.ox.ac.uk
Jane V
JV
Harper
Pamela
P
Reynolds
Emma L
EL
Leatherbarrow
Stanley W
SW
Botchway
Anthony W
AW
Parker
Peter
P
O'Neill
D000818:Animals; D002453:Cell Cycle / Q000528:radiation effects*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects*; D053904:DNA Breaks, Single-Stranded / Q000528:radiation effects; D004261:DNA Replication / Q000235:genetics* / Q000528:radiation effects*; D007700:Kinetics; D017785:Photons; D051135:Rad51 Recombinase / Q000378:metabolism
D004247:DNA; D051135:Rad51 Recombinase