The ATR-mediated S phase checkpoint prevents rereplication in mammalian cells when licensing control is disrupted.
DNA replication in eukaryotic cells is tightly controlled by a licensing mechanism, ensuring that each origin fires once and only once per cell cycle. We demonstrate that the ataxia telangiectasia and Rad3 related (ATR)-mediated S phase checkpoint acts as a surveillance mechanism to prevent rereplication. Thus, disruption of licensing control will not induce significant rereplication in mammalian cells when the ATR checkpoint is intact. We also demonstrate that single-stranded DNA (ssDNA) is the initial signal that activates the checkpoint when licensing control is compromised in mammalian cells. We demonstrate that uncontrolled DNA unwinding by minichromosome maintenance proteins upon Cdt1 overexpression is an important mechanism that leads to ssDNA accumulation and checkpoint activation. Furthermore, we show that replication protein A 2 and retinoblastoma protein are both downstream targets for ATR that are important for the inhibition of DNA rereplication. We reveal the molecular mechanisms by which the ATR-mediated S phase checkpoint pathway prevents DNA rereplication and thus significantly improve our understanding of how rereplication is prevented in mammalian cells.
Liu
Enbo
E
Department of Molecular Experimental Medicine and 2Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Lee
Alan Yueh-Luen
AY
Chiba
Takuya
T
Olson
Erin
E
Sun
Peiqing
P
Wu
Xiaohua
X
D000256:Adenoviridae / Q000235:genetics; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism / Q000494:pharmacology; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism; D016196:S Phase; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D051716:Replication Protein A; D016160:Retinoblastoma Protein; D025521:Tumor Suppressor Proteins; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases