Synthesis of DNA by DNA polymerase ε in vitro

S. L. Lee, Z. Q. Pan, A. D. Kwong, P. M.J. Burgers, J. Hurwitz

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167 Scopus citations

Abstract

The isolation of DNA polymerase (Pol) ε from extracts of HeLa cells is described. The final fractions contained two major subunits of 210 and 50 kDa which cosedimented with Polε activity, similar to those described previously (Syvaoja, J., and Linn, S. (1989) J. Biol. Chem. 264, 2489-2497). The properties of the human Polε and the yeast Polε were compared. Both enzymes elongated singly primed single-stranded circular DNA templates. Yeast Polε required the presence of a DNA binding protein (SSB) whereas human Polε required the addition of SSB, Activator 1 and proliferating cell nuclear antigen (PCNA) for maximal activity. Both enzymes were totally unable to elongate primed DNA templates in the presence of salt; however, activity could be restored by the addition of Activator 1 and PCNA. Like Polδ, Polε formed complexes with SSB-coated primed DNA templates in the presence of Activator 1 and PCNA which could be isolated by filtration through Bio-Gel A-5m columns. Unlike Polδ, Polε bound to SSB-coated primed DNA in the absence of the auxiliary factors. In the presence of salt, Polε complexes were less stable than they were in the absence of salt. In the in vitro simian virus 40 (SV40) T antigen-dependent synthesis of DNA containing the SV40 origin of replication, yeast Polε but not human Polε could substitute for yeast or human Polδ in the generation of long DNA products. However, human Polε did increase slightly the length of DNA chains formed by the DNA polymerase α-primase complex in SV40 DNA synthesis. The bearing of this observation on the requirement for a PCNA-dependent DNA polymerase in the synthesis and maturation of Okazaki fragments is discussed. However, no unique role for human Polε in the in vitro SV40 DNA replication system was detected.

Original languageEnglish
Pages (from-to)22707-22717
Number of pages11
JournalJournal of Biological Chemistry
Volume266
Issue number33
StatePublished - Jan 1 1991
Externally publishedYes

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    Lee, S. L., Pan, Z. Q., Kwong, A. D., Burgers, P. M. J., & Hurwitz, J. (1991). Synthesis of DNA by DNA polymerase ε in vitro. Journal of Biological Chemistry, 266(33), 22707-22717.