The human Tim/Tipin complex coordinates an Intra-S checkpoint response to UV that slows replication fork displacement.
UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m(2) UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.
Unsal-Kaçmaz
Keziban
K
Lineberger Comprehensive Cancer Center, CB 7295, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. wkarlk@med.unc.edu
Chastain
Paul D
PD
Qu
Ping-Ping
PP
Minoo
Parviz
P
Cordeiro-Stone
Marila
M
Sancar
Aziz
A
Kaufmann
William K
WK
D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D004247:DNA / Q000378:metabolism; D004261:DNA Replication / Q000528:radiation effects*; D004268:DNA-Binding Proteins; D015536:Down-Regulation / Q000528:radiation effects; D004789:Enzyme Activation / Q000528:radiation effects; D006367:HeLa Cells; D006801:Humans; D007065:Idoxuridine; D047908:Intracellular Signaling Peptides and Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000172:deficiency / Q000378:metabolism*; D011485:Protein Binding / Q000528:radiation effects; D011494:Protein Kinases / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism; D016196:S Phase / Q000528:radiation effects*; D014466:Ultraviolet Rays; D051760:Xeroderma Pigmentosum Group A Protein / Q000737:chemistry / Q000378:metabolism
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D051716:Replication Protein A; C509769:TIMELESS protein, human; C516227:Tipin protein, human; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D004247:DNA; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D007065:Idoxuridine