Organization of Replication Units and DNA Replication in Mammalian Cells as Studied by DNA Fiber Radioautography

Ляпунова, Н. А.

doi:10.1016/s0074-7696(08)62201-9

Cited by 19 publications

(20 citation statements)

References 108 publications

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“…In agreement with this model, recent studies of living cells expressing GFP-tagged PCNA by using the fluorescence redistribution after photobleaching (FRAP) assay (Axelrod et al, 1976;Lippincott-Schwartz et al, 2001) showed that focal GFP-PCNA exchanged very slowly (Sporbert et al, 2002). During S phase, immobile GFP-PCNA foci can assemble and disassemble in the nucleus with lifetimes ranging from 30 min to 3 h (Leonhardt et al, 2000), consistent with significant heterogeneity of replication units and the existence of very large replicons (Liapunova, 1994). However, another replication protein, GFP-RPA34, was found to be mobile at RFi and after bleaching completely redistributed within 2 min (Sporbert et al, 2002).…”

supporting

confidence: 53%

Section: Discussionmentioning

confidence: 99%

“…This may be caused by a slow exchange of PCNA in the moving replication complex with free PCNA molecules or by de novo assembly of PCNA clamps within a single large focus (Sporbert et al, 2002). The lifetime of GFP-PCNA foci in mid-late S-phase cells can reach 3 h (Leonhardt et al, 2000), comparable with the lifetime of mammalian replicons in mid-late S phase, which is ϳ3.5 h (Liapunova, 1994).Together, our results support a dynamic model of the normal replication complex in which relatively long-living and stable DNA-bound PCNA-based holocomplexes bind mobile subunits with low residence time comparable with the time of synthesis of one Okazaki fragment. Our results also directly support the concept of self-organization in cellular architecture (Misteli, 2001).…”

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confidence: 99%

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

Solovjeva

Svetlova

Sasina

et al. 2005

MBoC

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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...

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supporting

confidence: 53%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Solovjeva

Svetlova

Sasina

et al. 2005

MBoC

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“…DNA that is replicated late in S phase contains genes that are usually transcriptionally inactive and arranged into highly structured, heterochromatic regions that differ from the topologically more accessible, early replicating and transcriptionally active DNA (64). This topology might dictate different replication machinery so as to navigate this highly structured DNA but not having to navigate through transcribing DNA.…”

Section: Discussionmentioning

confidence: 99%

“…In fact, the rates of movement of replication forks differ, with late S phase supporting rates 2-3-fold faster than early S phase (reviewed in Ref. 64). A role for pol ⑀ in replication late in S phase would be consistent with the observation that temperature-sensitive mutants of the cat- alytic subunit of pol ⑀ in S. cerevisiae, when shifted to the non-permissive temperature, slow chromosomal DNA synthesis gradually until they arrest with 2C of DNA (9).…”

Section: Discussionmentioning

confidence: 99%

Human DNA Polymerase ε Colocalizes with Proliferating Cell Nuclear Antigen and DNA Replication Late, but Not Early, in S Phase

Fuss

Linn

2002

Journal of Biological Chemistry

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DNA polymerase ⑀ (pol ⑀) has been implicated in DNA replication, DNA repair, and cell cycle control, but its precise roles are unclear. When the subcellular localization of human pol ⑀ was examined by indirect immunofluorescence, pol ⑀ appeared in discrete nuclear foci that colocalized with proliferating cell nuclear antigen (PCNA) foci and sites of DNA synthesis only late in S phase. Early in S phase, pol ⑀ foci were adjacent to PCNA foci. In contrast to PCNA foci that were only present in S phase, pol ⑀ foci were present throughout mitosis and the G 1 phase of cycling cells. It is hypothesized from these observations that pol ⑀ and PCNA have separate but associated functions early in S phase and that pol ⑀ participates with PCNA in DNA replication late in S phase.Although as many as 13 DNA polymerases are known to exist in eukaryotes, only 3 are thought to be involved in chromosomal replication; they are DNA polymerases (pol) 1 ␣, ␦, and ⑀. pol ␣ is clearly involved in the synthesis and extension of RNA primers during DNA replication initiation (reviewed in Ref. 1), whereas pol ␦ is responsible for elongation, as most clearly demonstrated in SV40 DNA replication (2, 3). Although pol ⑀ is not required for SV40 replication (4, 5), it does appear to have a role in chromosomal DNA replication (5-10).DNA pol ⑀ is well conserved in sequence and subunit composition among eukaryotes. Human pol ⑀ has four subunits, a large catalytic subunit, p261, and three associated subunits, p59, p12, and p17 (11,12). Likewise, Saccharomyces cerevisiae pol ⑀ has a catalytic subunit of 256 kDa, POL2, and has three associated subunits encoded by DPB2, DPB3, DPB4 (8, 13-15).Both catalytic subunits are divided into two domains linked by a protease-sensitive region, an N-terminal domain containing polymerase and exonuclease motifs and a 120-kDa C-terminal domain. The S. cerevisiae and human catalytic subunits are 39% identical overall and have 63% identity in the N-terminal domain (16). p59 and DPB2p have 26% overall identity (17), whereas p17 and p12 have 36.5 and 34% identity to Dpb4p and Dpb3p, respectively (12). Given the conservation of pol ⑀ sequence and subunit structure between yeast and humans, it is likely that their functions are also largely conserved.A role for pol ⑀ in chromosomal DNA replication has been established in higher eukaryotes and in yeast. The elongation step of DNA replication is impaired in Xenopus laevis egg extracts immunodepleted of pol ⑀ (6). Additionally, using a polymerase trap technique that tags a polymerase with its DNA product, Zlotkin et al. (5) find that pol ⑀ from monkey kidney cells associates with nascent chromosomal DNA but not nascent SV40 DNA. Likewise, a neutralizing antibody against human pol ⑀ inhibits chromosomal replication when microinjected into human fibroblasts but fails to inhibit SV40 DNA replication in vitro (7). In S. cerevisiae, disruptions of POL2 are lethal and result in the terminal dumbbell morphology characteristic of a defect in DNA replication (8). When shifted to the no...

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DNA replication machinery of the mammalian cell

Malkas

1998

J. Cell. Biochem.

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Organization of Replication Units and DNA Replication in Mammalian Cells as Studied by DNA Fiber Radioautography

Cited by 19 publications

References 108 publications

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

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

Human DNA Polymerase ε Colocalizes with Proliferating Cell Nuclear Antigen and DNA Replication Late, but Not Early, in S Phase

DNA replication machinery of the mammalian cell

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