DNA polymerase clamp shows little turnover at established replication sites but sequential de novo assembly at adjacent origin clusters.
The spatial and temporal organization of DNA replication was investigated in living cells with a green fluorescent protein fusion to the DNA polymerase clamp PCNA. In situ extractions and photobleaching experiments revealed that PCNA, unlike RPA34, shows little if any turnover at replication sites, suggesting that it remains associated with the replication machinery through multiple rounds of Okazaki fragment synthesis. Photobleaching analyses further showed that the transition from earlier to later replicons occurs by disassembly into a nucleoplasmic pool of rapidly diffusing subcomponents and reassembly at newly activated sites. The fact that these replication sites were de novo assembled in close proximity to earlier ones suggests that activation of neighboring origins may occur by a domino effect possibly involving local changes in chromatin structure and accessibility.
Sporbert
Anje
A
Max Delbrück Center for Molecular Medicine, 13125 Berlin, Advanced Imaging Microscopy, 07745, Jena, Germany.
Gahl
Anja
A
Ankerhold
Richard
R
Leonhardt
Heinrich
H
Cardoso
M Cristina
MC
D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000235:genetics; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D032446:Myoblasts; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018741:Replication Origin; D015398:Signal Transduction
D002843:Chromatin; D008164:Luminescent Proteins; D018809:Proliferating Cell Nuclear Antigen; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004259:DNA-Directed DNA Polymerase