Checkpoint regulation of replication dynamics in UV-irradiated human cells.
At any moment during S phase, regions of genomic DNA are in various stages of replication (i.e., initiation, chain elongation, and termination). These stages may be differentially inhibited after treatment with various carcinogens that damage DNA such as UV. We used visualization of active replication units in combed DNA fibers, in combination with quantitative analyses of the size distributions of nascent DNA, to evaluate the role of S-checkpoint proteins in UV-induced inhibition of DNA replication. When HeLa cells were exposed to a low fluence (1 J/m(2)) of 254 nm UV light (UVC), new initiation events were severely inhibited (5-6-fold reduction). A larger fluence of UVC (10 J/m(2)) resulted in stronger inhibition of the overall rate of DNA synthesis without decreasing further the frequency of replicon initiation events. Incubation of HeLa cells with caffeine and knockdown of ATR or Chk1 kinases reversed the UVC-induced inhibition of initiation of new replicons. These findings illustrate the concordance of data derived from different experimental approaches, thus strengthening the evidence that the activation of the intra-S checkpoint by UVC is dependent on the ATR and Chk1 kinases.
Chastain
Paul D
PD
Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7525 USA. paul_chastain@med.unc.edu
Heffernan
Timothy P
TP
Nevis
Kathleen R
KR
Lin
Li
L
Kaufmann
William K
WK
Kaufman
David G
DG
Cordeiro-Stone
Marila
M
D064007:Ataxia Telangiectasia Mutated Proteins; D002110:Caffeine / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000235:genetics; D000071877:Checkpoint Kinase 1; D004247:DNA / Q000096:biosynthesis / Q000235:genetics / Q000528:radiation effects; D004249:DNA Damage / Q000235:genetics* / Q000528:radiation effects; D004261:DNA Replication / Q000235:genetics* / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D015536:Down-Regulation / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism* / Q000528:radiation effects; D018816:Genes, cdc / Q000502:physiology* / Q000528:radiation effects; D006367:HeLa Cells; D006801:Humans; D011494:Protein Kinases / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D016196:S Phase / Q000235:genetics* / Q000528:radiation effects; D014466:Ultraviolet Rays
D018797:Cell Cycle Proteins; D002110:Caffeine; D004247:DNA; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases