The fission yeast Cdc1 protein, a homologue of the small subunit of DNA polymerase delta, binds to Pol3 and Cdc27.

MacNeill, Stuart A.; Moreno, Sergio; Reynolds, Nicola; Nurse, Paul; Fantes, Peter A.

doi:10.1002/j.1460-2075.1996.tb00839.x

Cited by 96 publications

(146 citation statements)

References 53 publications

(17 reference statements)

Supporting

Mentioning

135

Contrasting

Order By: Relevance

“…Moreover, we have constructed a temperature-sensitive dpb2 mutant and found that these cells arrest in late G1/early S phase upon shift to the restrictive temperature. This phenotype has never been observed for temperature-sensitive mutants of either Pol ␣ or Pol ␦, suggesting that Pol ⑀ and Dpb2 provide a novel function(s) during DNA replication initiation (Hughes et al, 1992;D'Urso et al, 1995;MacNeill et al, 1996;Iino and Yamamoto, 1997;Reynolds et al, 1998;Reynolds and MacNeill, 1999). Consistent with this notion, using a chromatin immunoprecipitation (ChIP) assay, we find that both Dpb2 and Pol ⑀ bind replication origins early in S phase, very near to the time of DNA replication initiation.…”

Section: Introductionsupporting

confidence: 79%

“…Furthermore, we demonstrate that like cdc20 mutants (Feng and D'Urso, 2001), a temperature-sensitive mutant of dpb2 arrests with a 1C DNA content after shift to the restrictive temperature, suggesting that Dpb2 is required for the onset of S phase. Although it is currently impossible to rule out that DNA replication initiation occurs in cdc20 or dpb2 mutants, it is interesting that early S phase arrest phenotype has never been reported for mutants defective in other DNA polymerases or their associated subunits, all of which arrest with a near 2C DNA content (Hughes et al, 1992;Waseem et al, 1992;MacNeill et al, 1996;MacNeill and Fantes, 1997).Regardless of whether Pol ⑀ is directly involved in DNA replication initiation, it is clear from our studies that Cdc20 and Dpb2 provide an essential function that does not rely on their ability to synthesize DNA. One possibility is that Pol ⑀ binds replication origins early in S phase and is important for either recruiting or stabilizing the interaction of other replication proteins to DNA.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Section: Introductionsupporting

confidence: 79%

mentioning

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

show abstract

“…Mis6 and Mis12 code for proteins locating at the centromere (Saitoh et al 1997;Goshima et al 1999), while Mis4 is a sister chromatid cohesion molecule (Furuya et al 1998). Mis1, Mis5 and Mis10 are essential for replication (Takahashi et al 1994;MacNeill et al 1996; our unpublished results). These Mis gene products are probably implicated in maintaining the minichromosome through chromosome replication and separation.…”

Section: Introductionmentioning

confidence: 63%

Mis3 with a conserved RNA binding motif is essential for ribosome biogenesis and implicated in the start of cell growth and S phase checkpoint

2000

View full text Add to dashboard Cite

show abstract

“…Additionally, HYS2 mutants display supersensitivity to hydroxyurea, increased levels of mitotic chromosome loss, and recombination. More recently, MacNeill et al (23) cloned a Schizosaccharomyces pombe gene, cdc1 ϩ , which displays significant sequence identity (34%) to both the p50 subunit of pol ␦ and to HYS2. A physical interaction of the Cdc1 protein with the yeast pol ␦ protein was demonstrated by in vitro co-immunoprecipitation.…”

Section: Fig 10mentioning

confidence: 99%

Expression and Characterization of the Small Subunit of Human DNA Polymerase δ

Sun

Jiang

Zhang

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

DNA polymerase ␦ is a heterodimer consisting of a catalytic subunit of 125 kDa and a small subunit of 50 kDa (p50). We have overexpressed p50 in Escherichia coli and have characterized the recombinant protein. p50 was readily overexpressed using the pET vector as an insoluble protein. A procedure was developed for its purification and renaturation. Examination of the physicochemical properties of renatured p50 showed that it is a monomeric protein with an apparent molecular weight of 60,000, a Stokes radius of 34 Å, and a sedimentation coefficient of 4.1 S. Its physical properties were indistinguishable from p50 expressed as a soluble protein using the pTACTAC vector. Examination of the effects of recombinant p50 on the activity of DNA polymerase ␦ showed that p50 is able to slightly stimulate (about 2-fold) the activity of the recombinant 125-kDa catalytic subunit using poly(dA)⅐oligo(dT) as a template in the absence of proliferating cell nuclear antigen. In the presence of proliferating cell nulear antigen, activity is stimulated about 5-fold. Seven stable hybridoma cell lines were established that produced monoclonal antibodies against p50. One of these antibodies (13D5) inhibited the activity of calf thymus DNA polymerase ␦. This antibody, when coupled to a solid support, also was found to provide a method for the immunoafffinity purification of recombinant p50 and of DNA polymerase ␦ from calf thymus or HeLa extracts. Immunoprecipitation and enzyme-linked immunosorbent assays also confirmed that p50 interacts with the catalytic subunit of DNA polymerase ␦.DNA polymerase (pol) 1 ␦ is involved in both DNA replication (1) and DNA repair (2) in mammalian cells. Its activity is stimulated by PCNA, a DNA sliding clamp which provides the processivity required for its role in DNA replication (3, 4). In addition to PCNA, a number of other accessory proteins are required for the assembly of a functional replication complex at the leading strand of the replication fork. These include the multi-subunit replication factor C (RF-C) complex and replication protein A (RP-A), a single-stranded DNA-binding protein (5). pol ␦ isolated from calf thymus (6) and human placenta (7) is a heterodimer of 125-and 50-kDa polypeptides. The catalytic activity had been clearly demonstrated to be associated with the 125-kDa subunit (7), and preparations of mammalian pol ␦ have been reported that contain only the active 125-kDa subunit (8, 9). Goulian et al. (9) isolated two preparations of mouse DNA polymerase ␦, one of which consisted of a single polypeptide of 123-125 kDa and was unresponsive to PCNA stimulation. It was suggested that the 50-kDa polypeptide is required for the stimulation of DNA polymerase ␦ by PCNA (9). However, yeast pol ␦ catalytic subunit overexpressed in Escherichia coli was stimulated 2.5-to 3-fold (10). Human pol ␦ catalytic subunit expressed in the vaccinia virus system also showed a slight increase in activity in the presence of PCNA (11). This increase in activity is significantly lower than the 34-fold stimul...

show abstract

The fission yeast Cdc1 protein, a homologue of the small subunit of DNA polymerase delta, binds to Pol3 and Cdc27.

Cited by 96 publications

References 53 publications

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

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

Mis3 with a conserved RNA binding motif is essential for ribosome biogenesis and implicated in the start of cell growth and S phase checkpoint

Expression and Characterization of the Small Subunit of Human DNA Polymerase δ

Contact Info

Product

Resources

About