Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus

Weiser, Thomas; Gassmann, Max; Thömmes, Pia; Ferrari, Elena; Hafkemeyer, Peter; Hübscher, Ulrich

doi:10.1016/s0021-9258(18)99242-2

Cited by 135 publications

(30 citation statements)

References 59 publications

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“…3, lane 4). The pol δ antibody immunoblotted a band of 125 kDa, and the pol ε antibody detected a band of 145 kDa, in agreement with the previously reported molecular mass of pol ε isolated from calf thymus extracts (32,33). DNA polymerase α was not detected in the eluates by western blotting.…”

Section: Affinity Purification Of Dna Replication Complex On Pcna-sepharosesupporting

confidence: 90%

Identification of DNA replication and cell cycle proteins that interact with PCNA

Loor

Zhang

et al. 1997

Nucleic Acids Research

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The identity of DNA replication proteins and cell cycle regulatory proteins which can be found in complexes involving PCNA were investigated by the use of PCNA immobilized on Sepharose 4B. A column containing bovine serum albumin (BSA) bound to Sepharose was used as a control. Fetal calf thymus extracts were chromatographed on PCNA-Sepharose and BSA-Sepharose. The columns were washed and then eluted with 0.5 M KCl. The salt eluates were examined for the presence of both DNA replication proteins (Pol alpha, delta, straightepsilon, PCNA, RFC, RFA, DNA ligase I, NDH II, Topo I and Topo II) and cell cycle proteins (Cyclins A, B1, D1, D2, D3, E, CDK2, CDK4, CDK5 and p21) by western blotting with specific antibodies. The DNA replication proteins which bound to PCNA-Sepharose included DNA polymerase delta and straightepsilon, PCNA, the 37 and 40 kDa subunits of RFC, the 70 kDa subunit of RPA, NDH II and topoisomerase I. No evidence for the binding of DNA polymerase alpha, DNA ligase I or topoisomerase II was obtained. Of the cell cycle proteins investigated, CDK2, CDK4 and CDK5 were bound. This study presents strong evidence that PCNA is a component of protein complexes containing DNA replication, repair and cell cycle regulatory proteins.

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Section: Affinity Purification Of Dna Replication Complex On Pcna-sepharosesupporting

confidence: 90%

Identification of DNA replication and cell cycle proteins that interact with PCNA

Loor

Zhang

et al. 1997

Nucleic Acids Research

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“…Enzymes and Proteins. Pol R, pol δ, and RF-C were purified from fetal calf thymus as described (Weiser et al, 1991;Podust et al, 1992a). One unit of pol activity corresponds to the incorporation of 1 nmol of total dTMP into acid-precipitable material in 60 min at 37 °C in a standard assay containing 0.5 µg (nucleotides) of poly(dA)/ oligo(dT) 1:10 and 20 µM dTTP.…”

Section: Chemicalsmentioning

confidence: 99%

Phosphorylation of the PCNA Binding Domain of the Large Subunit of Replication Factor C by Ca²⁺/Calmodulin-Dependent Protein Kinase II Inhibits DNA Synthesis

et al. 1997

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Replication factor C (RF-C) is a heteropentameric protein essential for DNA replication and DNA repair. It is a molecular matchmaker required for loading of the proliferating cell nuclear antigen (PCNA) sliding clamp onto double-strand DNA and for PCNA-dependent DNA synthesis by DNA polymerases delta and epsilon. The DNA and PCNA binding domains of the large 140 kDa subunit of human RF-C have been recently cloned [Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Messier, H., Khastilba. S., Hübscher, U., & Fotedar, A. (1996) EMBO J. 15, 4423-4433]. Here we show that the PCNA binding domain is phosphorylated by the Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme required for cell cycle progression in eukaryotic cells. The DNA binding domain, on the other hand, is not phosphorylated. Phosphorylation by CaMKII reduces the binding of PCNA to RF-C and consequently inhibits RF-C-dependent DNA synthesis by DNA polymerases delta1 and epsilon. Once bound to PCNA and DNA, RF-C is protected from phosphorylation by CaMKII, suggesting a possible role of CaMKII in regulating the dynamics of interaction between PCNA and RF-C and thus interfering in the formation of an active sliding clamp by DNA polymerases delta and epsilon.

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“…Besides associating with and inhibiting cdk2 kinase (4)(5)(6)(7)(8), p21 has an additional activity through its interaction with the DNA replication factor PCNA. PCNA is an auxiliary factor for DNA polymerases δ and ε and is essential for DNA replication in vitro and in vivo (9)(10)(11)(12)(13)(14)(15). p21 interacts with PCNA and inhibits its activity (16)(17)(18)(19)(20)(21).…”

Section: Introductionmentioning

confidence: 99%

A 39 Amino Acid Fragment of the Cell Cycle Regulator p21 Is Sufficient to Bind PCNA and Partially Inhibit DNA Replication in vivo

Chen

Peters

Saha

et al. 1996

Nucleic Acids Research

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The cell cycle regulator p21 interacts with and inhibits the DNA replication and repair factor proliferating cell nuclear antigen (PCNA). We have defined a 39 amino acid fragment of p21 which is sufficient to bind PCNA with high affinity (Kd 10-20 nM). This peptide can inhibit DNA replication in vitro and microinjection of a GST fusion protein containing this domain inhibited S phase in vivo. Despite its high affinity for PCNA, the free 39 amino acid peptide does not have a well-defined structure, as judged from circular dichroism and nuclear magnetic resonance measurements, suggesting an induced fit mechanism for the PCNA-p21 interaction. The association of the small peptide with PCNA was thermolabile, suggesting that portions of p21 adjoining the minimal region of contact stabilize the interaction. In addition, a domain containing 67 amino acids from the N-terminus of PCNA was defined as both necessary and sufficient for binding to p21.

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Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus

Cited by 135 publications

References 59 publications

Identification of DNA replication and cell cycle proteins that interact with PCNA

Identification of DNA replication and cell cycle proteins that interact with PCNA

Phosphorylation of the PCNA Binding Domain of the Large Subunit of Replication Factor C by Ca²⁺/Calmodulin-Dependent Protein Kinase II Inhibits DNA Synthesis

A 39 Amino Acid Fragment of the Cell Cycle Regulator p21 Is Sufficient to Bind PCNA and Partially Inhibit DNA Replication in vivo

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Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus

Cited by 135 publications

References 59 publications

Identification of DNA replication and cell cycle proteins that interact with PCNA

Identification of DNA replication and cell cycle proteins that interact with PCNA

Phosphorylation of the PCNA Binding Domain of the Large Subunit of Replication Factor C by Ca2+/Calmodulin-Dependent Protein Kinase II Inhibits DNA Synthesis

A 39 Amino Acid Fragment of the Cell Cycle Regulator p21 Is Sufficient to Bind PCNA and Partially Inhibit DNA Replication in vivo

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Phosphorylation of the PCNA Binding Domain of the Large Subunit of Replication Factor C by Ca²⁺/Calmodulin-Dependent Protein Kinase II Inhibits DNA Synthesis