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Vol. 16, Issue 5, 2518-2528, May 2005

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High Mobility of Flap Endonuclease 1 and DNA Polymerase Associated with Replication Foci in Mammalian S-Phase Nucleus

Lioudmila Solovjeva ^*, Maria Svetlova ^*, Lioudmila Sasina , Kyoji Tanaka , Masafumi Saijo , Igor Nazarov , Morton Bradbury  ^||, and Nikolai Tomilin ^*

^* Laboratory of Chromosome Stability, Institute of Cytology, Russian Academy of Sciences, 194064 St. Petersburg, Russia; Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg, Russia; Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan; Department of Biochemistry and Molecular Medicine, School of Medicine, University of California, Davis, CA 95616; and ^|| Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM 87545

Submitted December 10, 2004; Revised February 15, 2005; Accepted February 21, 2005
Monitoring Editor: Joseph Gall

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 (Pol). 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 and GFP-Fen1 are mobile with residence times at RFi (t_m) 2 and 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 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.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Nikolai Tomilin (nvtom{at}mail.ru).

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