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

Abstract

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 ) ϳ2 and ϳ0.8 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. INTRODUCTIONIn mammalian cells, DNA replication during S phase is located at distinct nuclear sites. Replication foci (RFi) each contain on average 10 spatially clustered active replication forks that can be visualized using antibodies against incorporated halogenated deoxyuridines (Nakamura et al., 1986;Nakayasu and Berezney, 1989;van Dierendonck et al., 1989;O'Keefe et al., 1992;Tomilin et al., 1995;Jackson and Pombo, 1998). In fixed cells, the RFi colocalize with the Tritoninsoluble form of the replication protein proliferating cell nuclear antigen (PCNA) (Bravo and McDonald-Bravo, 1987), the p70 subunit of replication protein A (RPA70), protein kinase Cdk2 and cyclin A (Cardoso et al., 1993), DNA polymerase ␣ (Hozak et al., 1993), DNA ligase I (Cardoso et al., 1997), and DNA polymerase (Pol) (Kannouche et al., 2001). Distinct nuclear sites accumulate green fluorescent protein (GFP)-tagged DNA ligase I (Cardoso et al., 1997), GFP-PCNA (Leonhardt et al., 2000;Somanathan et al., 2001). The foci containing GFP-RPA34 (Sporbert et al., 2002) or GFP-Pol (Kannouche et al., 2001) also can be observed in normally proliferating living S-phase cells, indicating that the RFi seen in fixed cells do not arise during the fixation procedure but reflect real clustering of replication forks and associated molecular machines in the nucleus.It was initially suggested that the RFi represent specialized subnuclear organelles or nucleoskeleton-attached replication factories in which preassembled holocomplexes (replisomes) duplicate incoming DNA (Hozak et al., 1993). In agreement with this model, recent studies of living cells expressing GFP-tagged PCNA by using the fluorescence redistribution after photob...

Effect of Proliferating Cell Nuclear Antigen Ubiquitination and Chromatin Structure on the Dynamic Properties of the Y-family DNA Polymerases

“…The high mobility of pol in human cells, both uniformly distributed and in foci, agrees with the observations of Solovjeva et al (2005) using Chinese hamster cells, and emphasizes that even though visible in fluorescent replication structures, proteins may still interact there very transiently. Our biochemical data suggest that pol may be associated with another protein in a complex of total molecular mass of 112 kDa.…”

Elements That Regulate the DNA Damage Response of Proteins Defective in Cockayne Syndrome

“…The N-terminal GFP-CSA did localize to the nucleoplasm (but not the nucleolus), but was more prominently distributed to the cyctoplasm (Figure S5A). Conversely, the C-terminal GFP-CSA was primarily detected in the nucleoplasm, as would be expected for CSA based on prior reports 46, 49-51 , with little nucleolar localization (Figure S5A). Consistent with C-terminal GFP-CSA retaining normal folding and activity, we found that the fusion protein corrected the UV sensitivity of CS3BE CSA mutant cells (Figure S5B).…”

RAD18 and associated proteins are immobilized in nuclear foci in human cells entering S-phase with ultraviolet light-induced damage

“…This would be accomplished by recruitment mechanisms. Indeed, the residence time of polη in replication foci of cells that have not been damaged is very short [30]. …”