A novel family of DNA-polymerase-associated B subunits

Mäkiniemi, Minna; Pospiech, Helmut; Kilpeläinen, Simo; Jokela, Maarit; Vihinen, Mauno; Syväoja, Juhani E.

doi:10.1016/s0968-0004(98)01327-9

Cited by 58 publications

(43 citation statements)

References 22 publications

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“…To select a target area for mutagenesis, we compared all known Dpb2 proteins to identify conserved amino acid residues. As previously reported, the regulatory B-type subunits of eukaryotic DNA polymerases, which includes Dpb2, belong to a protein superfamily (Makiniemi et al, 1999) that contains a calcineurin-like phosphatase domain similar to those found in archaeal X-type DNA polymerases (Aravind and Koonin, 1999). Although some of the key residues required for phosphatase activity are no longer present in the eukaryotic proteins (Aravind and Koonin, 1999), some of the remaining conserved residues might be important to stabilize the protein.…”

Section: A Temperature-sensitive Dpb2 Mutant Arrests In Early S Phasementioning

confidence: 77%

“…Although some of the key residues required for phosphatase activity are no longer present in the eukaryotic proteins (Aravind and Koonin, 1999), some of the remaining conserved residues might be important to stabilize the protein. In fact, several temperature-sensitive mutations found in eukaryotic DNA polymerase B-type subunits have been localized to this region of the protein (Makiniemi et al, 1999). For example, in the HYS2/POL31 gene of S. cerevisiae, a mutation converting an Asp to Asn at amino acid position 304 renders the protein temperature sensitive (Hashimoto et al, 1998).…”

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confidence: 99%

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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

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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...

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Section: A Temperature-sensitive Dpb2 Mutant Arrests In Early S Phasementioning

confidence: 77%

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

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“…All four catalytic subunits possess two putative zinc ®nger modules close to their C-termini, and genetic analyses in budding yeast have shown that mutations within these modules abolish polymerase function in vivo, at least in the cases of Pol d and Pol e (4,18). Amongst the non-catalytic subunits, only the Bsubunits of Pol a, Pol d and Pol e are related to one another (34). In budding yeast, each B-subunit (Pol12, Pol31 or Dpb2) is essential for complete chromosomal DNA replication and, therefore, for cell viability (3).…”

Section: Discussionmentioning

confidence: 99%

The C-terminal zinc finger of the catalytic subunit of DNA polymerase is responsible for direct interaction with the B-subunit

García

Ciufo²,

Yang³

et al. 2004

Nucleic Acids Research

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DNA polymerase d (Pol d) plays a central role in eukaryotic chromosomal DNA replication, repair and recombination. In ®ssion yeast, Pol d is a tetrameric enzyme, comprising the catalytic subunit Pol3 and three smaller subunits, Cdc1, Cdc27 and Cdm1. Previous studies have demonstrated a direct interaction between Pol3 and Cdc1, the B-subunit of the complex. Here it is shown that removal of the tandem zinc ®nger modules located at the C-terminus of Pol3 by targeted proteolysis renders the Pol3 protein non-functional in vivo, and that the Cterminal zinc ®nger module ZnF2 is both necessary and suf®cient for binding to the B-subunit in vivo and in vitro. Extensive mutagenesis of the ZnF2 module identi®es important residues for B-subunit binding. In particular, disruption of the ZnF2 module by substitution of the putative metal-coordinating cysteines with alanine abolishes B-subunit binding and in vivo function. Finally, evidence is presented suggesting that the ZnF region is post-translationally modi®ed in ®ssion yeast cells.

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“…No enzymatic activity has been assigned to this polypeptide, which has been implicated in the stabilization and regulation of the catalytic subunit Pol1. The B subunit is common to most eukaryotic DNA polymerases and shows several conserved motifs, particularly in the C-terminal portion of the protein (Collins et al 1993;Makiniemi et al 1999).…”

Section: The Pol12 Gene Plays a Role In Telomere Length Regulationmentioning

confidence: 99%

“…Although the pol12-216 mutation falls in a short nonconserved region, it might be significant that this stretch of amino acids, located between two conserved sequences, appears to be an ∼6 amino acid insertion found only in the B subunits of Pol␣-primase complexes, from yeast to humans (Makiniemi et al 1999). pol12-216 defines a distinct telomere function required for viability in cells with reduced STN1 activity Several lines of evidence strongly suggest that the telomere alteration in pol12-216 cells is distinct from that caused by hypomorphic ts-lethal alleles of POL1, and is not caused by a general defect in lagging-strand DNA synthesis.…”

Section: The Pol12 Gene Plays a Role In Telomere Length Regulationmentioning

confidence: 99%

Pol12, the B subunit of DNA polymerase α, functions in both telomere capping and length regulation

Grossi¹,

Puglisi²,

Dmitriev³

et al. 2004

Genes Dev.

129

173

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The regulation of telomerase action, and its coordination with conventional DNA replication and chromosome end "capping," are still poorly understood. Here we describe a genetic screen in yeast for mutants with relaxed telomere length regulation, and the identification of Pol12, the B subunit of the DNA polymerase ␣ (Pol1)-primase complex, as a new factor involved in this process. Unlike many POL1 and POL12 mutations, which also cause telomere elongation, the pol12-216 mutation described here does not lead to either reduced Pol1 function, increased telomeric single-stranded DNA, or a reduction in telomeric gene silencing. Instead, and again unlike mutations affecting POL1, pol12-216 is lethal in combination with a mutation in the telomere end-binding and capping protein Stn1. Significantly, Pol12 and Stn1 interact in both two-hybrid and biochemical assays, and their synthetic-lethal interaction appears to be caused, at least in part, by a loss of telomere capping. These data reveal a novel function for Pol12 and a new connection between DNA polymerase ␣ and Stn1. We propose that Pol12, together with Stn1, plays a key role in linking telomerase action with the completion of lagging strand synthesis, and in a regulatory step required for telomere capping.

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A novel family of DNA-polymerase-associated B subunits

Cited by 58 publications

References 22 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

The C-terminal zinc finger of the catalytic subunit of DNA polymerase is responsible for direct interaction with the B-subunit

Pol12, the B subunit of DNA polymerase α, functions in both telomere capping and length regulation

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