Abstract
Replication of singly-DNA primed M13 DNA by DNA polymerase (pol) δ completely relies on the simultaneous addition of proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) (orE.coli singlestrand DNA binding protein, SSB). Pol ɛ core alone is able to synthesize the products on singly-primed ssDNA. However, DNA synthesis by pol ɛ was stimulated up to 10-fold upon addition of the three auxiliary proteins PCNA, RF-C and SSB. This stimulation of pol ɛ by PCNA/RF-C/SSB appears to be the superposition of two events: pol, ɛ holoenzyme (pol ɛ, PCNA, RF-C) synthesized longer products than its pol ɛ core counterpart, but elongated less primers. Furthermore, we analyzed the cooperative action of pol α/primase with pol δ or pol ɛ holoenzymes on unprimed M13 DNA. While pol δ displayed higher dNMP incorporation than pol ɛ, when a single primer was preannealed to DNA, pol ɛ was more efficient in the utilization of the primers synthesized by pol α/primase. Under these conditions both longer products and a higher amount of dNMP incorporation was found for pol ɛ holoenzyme, than for pol δ. Our data support the hypothesis of pol δ as the leading and pol ɛ as the second lagging strand replication enzyme.
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Abbreviations
- pol:
-
DNA polymerase
- PCNA:
-
proliferating cell nuclear antigen
- RP-A:
-
replication protein A
- RF-C:
-
replication factor C
- SSB:
-
single-strand DNA binding protein
- ssDNA:
-
single-strand DNA
- SV40:
-
simian virus 40
- T antigen:
-
SV 40 large tumor antigen
- DTT:
-
dithiothreitol
- BSA:
-
bovine serum albumin
References
Alberts BM (1987) Prokaryotic DNA replication mechanisms. Phil Trans R Soc Lond [Biol] 317: 395–420
Burgers PMJ (1988) Mammalian cyclin/PCNA (DNA polymerase δ auxiliary protein) stimulates processive DNA synthesis by yeast DNA polymerase III. Nucleic Acids Res. 16: 6297–6307
Burgers PMJ, Bambara RA, Campbell JL Chang LMS, Downey KM, Hübscher U, Lee MYWT, Linn S, So AG, Spadari S (1990) Revised nomenclature for eukaryotic DNA polymerases. Eur J Biochem 191 617–618
Burgers PMJ (1991)Saccharomyces cerevisiae replication factor C. II. Formation and activity of complexes with proliferating cell nuclear antigen and with DNA polymerases δ and ɛ. J Biol Chem 266: 22698–22706
Chang LMS, Rafter E, Augl C, Bollum FJ (1984) Purification of a DNA polymerase-DNA primase complex from calf thymus glands. J Biol Chem 259: 14679–14687
Chen M, Pan Z-Q, Hurwitz J (1992a) Sequence and expression inEscherichia coli of the 40-kDa subunit of activator 1 (replication factor C) of Hela cells. Proc Natl Acad Sci USA 89: 2516–2520
Chen M, Pan Z-Q, Hurwitz J (1992b) Studies of the cloned 37-kDa subunit of activator 1 (replication factor C) of Hela cells. Proc Natl Acad Sci USA 89: 5211–5215
Dornreiter I, Erdile LF, Gilbert IU, Winkler D, Kelly TJ, Fanning E (1992) Interaction of DNA polymerase α with cellular replication protein A and SV40 T antigen. EMBO J 11: 769–776
Dutta A, Stillman B (1992)cdc2family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication. EMBO J 11: 2189–2199
Fien K, Stillman B (1992) Identification of replication factor C fromSaccharomyces cerevisiae: a component of the leading strand DNA replication complex. Mol Cell Biol 12: 155–163
Focher F, Gassmann M, Hafkemyer P, Ferrari E, Spadari S, Hübscher U (1989) Calf thymus DNA polymerase δ independent of proliferating cell nuclear antigen (PCNA). Nucleic Acids Res 17: 1805–1821
Georgaki A, Strack B, Podust V, Hübscher U (1992) DNA unwinding activity of replication protein A. FEBS Lett 308: 240–244
Hübscher U, Thömmes P (1992) DNA polymerase ɛ: in search of a function. Trends Biochem Sci 17: 55–58
Kenny MK, Lee S-H, Hurwitz J (1989) Multiple functions of human single-stranded-DNA binding protein in simian virus 40 DNA replication: single-strand stabilization and stimulation of DNA polymerases α and δ. Proc Natl Acad Sci USA 86: 9757–9761
Kim C, Snyder RO, Wold MS (1992) Binding properties of re-plication protein A from human and yeast cells. Mol Cell Biol 12: 3050–3059
Kornberg A, Baker TA (1992) DNA replication. W.H. Freeman, New York
Lee S-H, Hurwitz J (1990) Mechanism of elongation of primed DNA by DNA polymerase δ, proliferating cell nuclear antigen, and activator 1. Proc Natl Acad Sci USA 87: 5672–5676
Lee S-H, Kwong AD, Pan Z-Q, Hurwitz J (1991a) Studies on the activator 1 protein complex, an accessory factor for proliferating cell nuclear antigen-dependent DNA polymerase δ. J Biol Chem 266: 594–602
Lee S-H, Pan Z-Q, Kwong AD, Burgers PMJ, Hurwitz J (1991b) Synthesis of DNA by DNA polymerase ɛin vitro. J Biol Chem 266: 22707–22717
Loman TM, Green JM, Beyer RS (1986) Large scale overproduction and rapid purification of theEscherichia coli ssb gene product. Expression of thessb gene under λ PL control. Biochemistry 25: 21–25
Maki H, Maki S, Kornberg A (1988) DNA polymerase III holoenzyme ofEscherichia coli. The holoenzyme is an asymmetric dimer with twin active sites. J Biol Chem 263: 6570–6578
McHenry CS (1991) DNA polymerase III holoenzyme. Components, structure, and mechanism of a true replicative complex. J Biol Chem 266: 19127–19130
Nasheuer H-P, Grosse F (1987) Immunoaffinity-purified DNA polymerase α displays novel properties. Biochemistry 26: 8458–8466
Nethanel T, Kaufmann G (1990) Two DNA polymerases may be required for synthesis of the lagging DNA strand of simian virus 40. J Virol 64: 5912–5918
Podust VN, Georgaki A, Strack B, Hübscher U (1992) Calf thymus RF-C as an essential component for DNA polymerase δ and ɛ holoenzymes function. Nucleic Acids Res 20: 4159–4165
Prelich G, Tan C-K, Kostura M, Mathews MB, So AG, Downey KM, Stillman B (1987) The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replicationin vitro. Nature 326: 517–520
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Thömmes P, Hübscher U (1990) Eukaryotic DNA replication. Enzymes and proteins acting at the fork. Eur J Biochem 194: 699–712
Tsurimoto T, Stillman B (1989a) Purification of a cellular replication factor, RF-C, that is required for coordinated synthesis of leading and lagging strands during simian virus 40 DNA re-plication in vitro. Mol Cell Biol 9: 609–619
Tsurimoto T, Stillman B (1989b) Multiple replication factors augment DNA synthesis by the two eukaryotic DNA poly-merases, α and δ. EMBO J. 8: 3883–3889
Tsurimoto T, Stillman B (1990) Functions of replication factor C and proliferating-cell nuclear antigen: functional similarity of DNA accessory proteins from human cells and bacteriophage T4. Proc Natl Acad Sci USA 87: 1023–1027
Tsurimoto T, Stillman B (1991a) Replication factors required for SV40 DNA replicationin vitro. I. DNA structure-specific recognition of a primer-template junction by eukaryotic DNA polymerases and their accessory proteins. J Biol Chem 266: 1950–1960
Tsurimoto T, Stillman B (1991b) Replication factors required for SV40 DNA replicationin vitro. II. Switching of DNA polymerase α and δ during initiation of leading and lagging strand synthesis. J Biol Chem 266: 1961–1968
Wang TS-F (1991) Eukaryotic DNA polymerases. Annu Rev Biochem 60: 513–552
Weiser T, Gassmann M, Thömmes P, Ferrari E, Hafkemeyer P, Hübscher U (1991) Biochemical and functional comparison of DNA polymerases α, δ, and ɛ from calf thymus. J Biol Chem 266: 10420–10428
Wold MS, Kelly T (1988) Purification and characterization of replication protein A, a cellular protein required forin vitro replication of simian virus 40 DNA. Proc Natl Acad Sci USA 85: 2523–2527
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On sabbatical leave from Kol'tzov Institute of Developmental Biology, 117334, Moscow, Russia
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Podust, V., Mikhailov, V., Georgaki, A. et al. DNA polymerase δ and ɛ holoenzymes from calf thymus. Chromosoma 102 (Suppl 1), S133–S141 (1992). https://doi.org/10.1007/BF02451797
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DOI: https://doi.org/10.1007/BF02451797