Structure and function of the fourth subunit (Dpb4p) of DNA polymerase varepsilon in Saccharomyces cerevisiae

Ohya, Tomoko; Maki, Satoko; Kawasaki, Yasuo; Sugino, Akio

doi:10.1093/nar/28.20.3846

Cited by 70 publications

(77 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Therefore, p17 might mediate an interaction or a switching between pol ⑀ and huCHRAC to facilitate efficient replication of the highly ordered heterochromatin. Deletion mutants of DPB4, the S. cerevisiae homolog of human pol ⑀ p17, are viable but have a prolonged S-phase (15), supporting the proposal that pol ⑀ might be necessary for the efficient replication of heterochromatic DNA though another polymerase (presumably pol ␦), and other chromatin-remodeling mechanisms can operate in its place.…”

Section: Discussionmentioning

confidence: 62%

See 1 more Smart Citation

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

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionmentioning

confidence: 62%

“…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)(14)(15).…”

mentioning

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

View full text Add to dashboard Cite

show abstract

“…DPB3 and DPB4 encode the two nonessential components of DNA polymerase e (Pol e) and have been implicated in replication fork progression (Araki et al 1991;Dua et al 1998;Ohya et al 2000). In addition, previous studies have demonstrated roles for Dpb3 and Dpb4 in telomeric silencing (Tsubota et al 2006) and have shown that Dpb4 is also a component of the Isw2 complex (Iida and Araki 2004;McConnell et al 2004).…”

Section: Resultsmentioning

confidence: 99%

Alterations in DNA Replication and Histone Levels Promote Histone Gene Amplification in Saccharomyces cerevisiae

Libuda¹,

Winston

2010

Genetics

View full text Add to dashboard Cite

Gene amplification, a process that increases the copy number of a gene or a genomic region to two or more, is utilized by many organisms in response to environmental stress or decreased levels of a gene product. Our previous studies in Saccharomyces cerevisiae identified the amplification of a histone H2A-H2B gene pair, HTA2-HTB2, in response to the deletion of the other H2A-H2B gene pair, HTA1-HTB1. This amplification arises from a recombination event between two flanking Ty1 elements to form a new, stable circular chromosome and occurs at a frequency higher than has been observed for other Ty1-Ty1 recombination events. To understand the regulation of this amplification event, we screened the S. cerevisiae nonessential deletion set for mutations that alter the amplification frequency. Among the deletions that increase HTA2-HTB2 amplification frequency, we identified those that either decrease DNA replication fork progression (rrm3D, dpb3D, dpb4D, and clb5D) or that reduce histone H3-H4 levels (hht2-hhf2D). These two classes are related because reduced histone H3-H4 levels increase replication fork pauses, and impaired replication forks cause a reduction in histone levels. Consistent with our mutant screen, we found that the introduction of DNA replication stress by hydroxyurea induces the HTA2-HTB2 amplification event. Taken together, our results suggest that either reduced histone levels or slowed replication forks stimulate the HTA2-HTB2 amplification event, contributing to the restoration of normal chromatin structure.

show abstract

“…Only Dpb2 is essential for viability and normal S phase progression (Araki et al, 1991a). Although both Dpb3 and Dpb4 are not required for cell viability, deletion of the genes encoding these proteins does result in partial defects in DNA replication (Araki et al, 1991b;Ohya et al, 2000). Interestingly, both Dpb3 and Dpb4 contain histone-fold motifs, suggesting these proteins might be involved in chromatin remodeling (Araki et al, 1991b;Li et al, 2000;Ohya et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

“…Although both Dpb3 and Dpb4 are not required for cell viability, deletion of the genes encoding these proteins does result in partial defects in DNA replication (Araki et al, 1991b;Ohya et al, 2000). Interestingly, both Dpb3 and Dpb4 contain histone-fold motifs, suggesting these proteins might be involved in chromatin remodeling (Araki et al, 1991b;Li et al, 2000;Ohya et al, 2000). Based on the existence of these motifs, it has been proposed these subunits might facilitate replication through heterochromatic regions of DNA (Fuss and Linn, 2002).…”

Section: Introductionmentioning

confidence: 99%

Schizosacchromyces pombeDpb2 Binds to Origin DNA Early in S Phase and Is Required for Chromosomal DNA Replication

Feng

Rodriguez-Menocal

Tolun

et al. 2003

MBoC

View full text Add to dashboard Cite

Genetic evidence suggests that DNA polymerase epsilon (Pol ⑀) has a noncatalytic essential role during the early stages of DNA replication initiation. Herein, we report the cloning and characterization of the second largest subunit of Pol ⑀ in fission yeast, called Dpb2. We demonstrate that Dpb2 is essential for cell viability and that a temperature-sensitive mutant of dpb2 arrests with a 1C DNA content, suggesting that Dpb2 is required for initiation of DNA replication. Using a chromatin immunoprecipitation assay, we show that Dpb2, binds preferentially to origin DNA at the beginning of S phase. We also show that the C terminus of Pol ⑀ associates with origin DNA at the same time as Dpb2. We conclude that Dpb2 is an essential protein required for an early step in DNA replication. We propose that the primary function of Dpb2 is to facilitate assembly of the replicative complex at the start of S phase. These conclusions are based on the novel cell cycle arrest phenotype of the dpb2 mutant, on the previously uncharacterized binding of Dpb2 to replication origins, and on the observation that the essential function of Pol ⑀ is not dependent on its DNA synthesis activity. INTRODUCTIONChromosomal DNA replication in eukaryotic cells requires the activity of at least three DNA polymerases: ␣, ␦, and ⑀ (Waga and Stillman, 1998;Hubscher et al., 2000;Bell and Dutta, 2002). Pol ␣ is tightly associated with a primase activity capable of de novo DNA synthesis, suggesting that this enzyme is responsible for initiation of both leading and lagging strands (Waga and Stillman, 1998). Pol ␣/primase can only synthesize short primers that must then be extended by the activity of a processive DNA polymerase(s). Biochemical analysis of simian virus 40 (SV40) DNA replication, which has been extensively used as a model system for eukaryotic DNA replication, has shown that primers synthesized by Pol ␣ can be extended by Pol ␦ and that these two polymerases are sufficient for SV40 replication in vitro (Weinberg et al., 1990;Waga et al., 1994).A second processive DNA polymerase, Pol ⑀, is required for cell viability and chromosomal DNA replication in both fission (D'Urso and Nurse, 1997) and budding yeast (Morrison et al., 1990;Araki et al., 1992;Budd and Campbell, 1993). Pol ⑀ has also been implicated in both DNA repair (Jessberger et al., 1993;Wang et al., 1993;Shivji et al., 1995;Holmes, 1999) and cell cycle checkpoint control in eukaryotic cells (Navas et al., 1995). Recently, we have shown that the DNA polymerase and exonuclease domains of Pol ⑀ are dispensable for cell viability in fission yeast (Feng and D'Urso, 2001). Similar observations have been made for the evolutionarily distant yeast, Saccharomyces cerevisiae (Kesti et al., 1999;Dua et al., 2000). These findings have raised important questions regarding the essential role of this replicative enzyme in DNA synthesis. Although the N-terminal catalytic domains are dispensable, the C-terminal half of the enzyme is essential for cell viability and chromosomal replication in fission...

show abstract

Structure and function of the fourth subunit (Dpb4p) of DNA polymerase varepsilon in Saccharomyces cerevisiae

Cited by 70 publications

References 35 publications

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

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

Alterations in DNA Replication and Histone Levels Promote Histone Gene Amplification in Saccharomyces cerevisiae

Schizosacchromyces pombeDpb2 Binds to Origin DNA Early in S Phase and Is Required for Chromosomal DNA Replication

Contact Info

Product

Resources

About