Abstract
The isolation of DNA polymerase (Pol) epsilon from extracts of HeLa cells is described. The final fractions contained two major subunits of 210 and 50 kDa which cosedimented with Pol epsilon activity, similar to those described previously (Syvaoja, J., and Linn, S. (1989) J. Biol. Chem. 264, 2489-2497). The properties of the human Pol epsilon and the yeast Pol epsilon were compared. Both enzymes elongated singly primed single-stranded circular DNA templates. Yeast Pol epsilon required the presence of a DNA binding protein (SSB) whereas human Pol epsilon 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 Pol delta, Pol epsilon 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 Pol delta, Pol epsilon bound to SSB-coated primed DNA in the absence of the auxiliary factors. In the presence of salt, Pol epsilon 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 Pol epsilon but not human Pol epsilon could substitute for yeast or human Pol delta in the generation of long DNA products. However, human Pol epsilon did increase slightly the length of DNA chains formed by the DNA polymerase alpha-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 Pol epsilon in the in vitro SV40 DNA replication system was detected.
Highlights
Synthesis of DNA by DNA Polymerase E in Vitro*The properties of the human Pole and the yeast Pole were compared
If complex with the Al-PCNA-primed DNA complex which is such a complex were to exist, thiscould position Polt with or not stable to the gel filtration step. These findings suggest near Polawhich could facilitate thecombined action of these that during elongation reactions, the processivities of Pole polymerases in lagging strand synthesis.Based on the studies and Po16 may differ
Because multisubunit SSBs (HSSB andYSSB) showed some specificity in stimulating hPok activity on a primed DNA (Fig. 6), it is possible that hPolt functions as a repair polymerase
Summary
The properties of the human Pole and the yeast Pole were compared Both enzymes elongated singly primed single-stranded circular DNA templates. DNA primase catalyzes the initiation and synthesis ofoligoribonucleotides which are elongated immediately by Pola These DNA products (Okazaki fragments) from the lagging strand template. The second enzyme, DNA polymerase 6 (Po16)consists of a catalytic subunitof 125 kDa arise erating cell nuclear antigen(PCNA)for maximal activ- and another subunit of 50 kDa of unknown function (Lee et ity.Bothenzymes were totallyunabletoelongate al., 1984; Goulian et al, 1990). In the presence of PCNA and primed DNA templates in the presence of salt; how- a multisubunit complex called Activator I ( A l ) or replication ever, activity could be restored by the addition of Ac- factor C (RF C), Po16 catalyzes the elongation of Okazaki tivator 1and PCNA.
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