Viral transport of DNA damage that mimics a stalled replication fork.
Adeno-associated virus type 2 (AAV2) infection incites cells to arrest with 4N DNA content or die if the p53 pathway is defective. This arrest depends on AAV2 DNA, which is single stranded with inverted terminal repeats that serve as primers during viral DNA replication. Here, we show that AAV2 DNA triggers damage signaling that resembles the response to an aberrant cellular DNA replication fork. UV treatment of AAV2 enhances the G2 arrest by generating intrastrand DNA cross-links which persist in infected cells, disrupting viral DNA replication and maintaining the viral DNA in the single-stranded form. In cells, such DNA accumulates into nuclear foci with a signaling apparatus that involves DNA polymerase delta, ATR, TopBP1, RPA, and the Rad9/Rad1/Hus1 complex but not ATM or NBS1. Focus formation and damage signaling strictly depend on ATR and Chk1 functions. Activation of the Chk1 effector kinase leads to the virus-induced G2 arrest. AAV2 provides a novel way to study the cellular response to abnormal DNA replication without damaging cellular DNA. By using the AAV2 system, we show that in human cells activation of phosphorylation of Chk1 depends on TopBP1 and that it is a prerequisite for the appearance of DNA damage foci.
Jurvansuu
Jaana
J
Swiss Institute for Experimental Cancer Research and National Center of Competence in Research Molecular Oncology, Epalinges, Lausanne, Switzerland.
Raj
Kenneth
K
Stasiak
Andrzej
A
Beard
Peter
P
D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis; D004268:DNA-Binding Proteins; D000229:Dependovirus / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology / Q000528:radiation effects; D016195:G2 Phase; D006801:Humans; D008954:Models, Biological; D009687:Nuclear Proteins; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D014466:Ultraviolet Rays
D002352:Carrier Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C110513:TOPBP1 protein, human; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1