Uncoupling the replication machinery: replication fork progression in the absence of processive DNA synthesis.
The precise coordination of the different steps of DNA replication is critical for the maintenance of genome stability. We have probed the mechanisms coupling various components of the replication machinery and their response to polymerase stalling by inhibition of the DNA polymerases in living mammalian cells with aphidicolin. We observed little change in the behaviour of proteins involved in the initiation of DNA replication. In contrast, we detected a marked accumulation of the single stranded DNA binding factor RPA34 at sites of DNA replication. Finally, we demonstrate that proteins involved in the elongation step of DNA synthesis dissociate from replication foci in the presence of aphidicolin. Taken together, these data indicate that inhibition of processive DNA polymerases uncouples the initiation of DNA replication from subsequent elongation steps. We, therefore, propose that the replication machinery is made up of distinct functional sub-modules that allow a flexible and dynamic response to challenges during DNA replication.
Görisch
Sabine M
SM
Max Delbrück Center for Molecular Medicine, Berlin, Germany.
Sporbert
Anje
A
Stear
Jeffrey H
JH
Grunewald
Ingrid
I
Nowak
Danny
D
Warbrick
Emma
E
Leonhardt
Heinrich
H
Cardoso
M Cristina
MC
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D018809:Proliferating Cell Nuclear Antigen; D012097:Repressor Proteins; D016590:Aphidicolin; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D011088:DNA Ligases; D000072481:DNA Ligase ATP