High mobility of flap endonuclease 1 and DNA polymerase eta associated with replication foci in mammalian S-phase nucleus.
Originally detected in fixed cells, DNA replication foci (RFi) were later visualized in living cells by using green fluorescent protein (GFP)-tagged proliferating cell nuclear antigen (PCNA) and DNA ligase I. It was shown using fluorescence redistribution after photobleaching (FRAP) assay that focal GFP-PCNA slowly exchanged, suggesting the existence of a stable replication holocomplex. Here, we used the FRAP assay to study the dynamics of the GFP-tagged PCNA-binding proteins: Flap endonuclease 1 (Fen1) and DNA polymerase eta (Pol eta). We also used the GFP-Cockayne syndrome group A (CSA) protein, which does associate with transcription foci after DNA damage. In normal cells, GFP-Pol eta and GFP-Fen1 are mobile with residence times at RFi (t(m)) approximately 2 and approximately 0.8 s, respectively. GFP-CSA is also mobile but does not concentrate at discrete foci. After methyl methanesulfonate (MMS) damage, the mobile fraction of focal GFP-Fen1 decreased and t(m) increased, but it then recovered. The mobilities of focal GFP-Pol eta and GFP-PCNA did not change after MMS. The mobility of GFP-CSA did not change after UV-irradiation. These data indicate that the normal replication complex contains at least two mobile subunits. The decrease of the mobile fraction of focal GFP-Fen1 after DNA damage suggests that Fen1 exchange depends on the rate of movement of replication forks.
Solovjeva
Lioudmila
L
Laboratory of Chromosome Stability, Institute of Cytology, Russian Academy of Sciences, 194064 St. Petersburg, Russia.
Svetlova
Maria
M
Sasina
Lioudmila
L
Tanaka
Kyoji
K
Saijo
Masafumi
M
Nazarov
Igor
I
Bradbury
Morton
M
Tomilin
Nikolai
N
D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active / Q000187:drug effects / Q000528:radiation effects; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D004249:DNA Damage; D045643:DNA Repair Enzymes; D004261:DNA Replication; D018076:DNA, Complementary / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D045585:Flap Endonucleases / Q000235:genetics / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008741:Methyl Methanesulfonate / Q000633:toxicity; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016196:S Phase / Q000502:physiology*; D014157:Transcription Factors; D014162:Transfection; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
D045643:DNA Repair Enzymes; D018076:DNA, Complementary; D004259:DNA-Directed DNA Polymerase; D045585:Flap Endonucleases; D049452:Green Fluorescent Proteins; D008741:Methyl Methanesulfonate; D018809:Proliferating Cell Nuclear Antigen; D011506:Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C095472:ERCC8 protein, human; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases; C468317:FEN1 protein, human