Comparison of DNA polymerases α and δ from bone marrow

Abstract

DNA polymerases alpha and delta from rabbit bone marrow were purified to specific activities greater than 30 000 nM TMP incorporated (mg of protein)(-1)h(-1). alpha is quantitatively predominant. alpha and delta have the same reaction requirements and are both similarly sensitive to N-ethylmaleimide. The primary functional distinction is the association of 3' to 5' exonuclease activity with delta. Sedimentation coefficients obtained from zone sedimentation in glycerol gradients and Stokes radii values from gel filtration allow the calculation of true molecular weight and frictional ratios. alpha exhibits a bimodal pattern, sedimenting at 6 and 8 S on glycerol gradients and demonstrating components corresponding to 40.5- and 65-A Stokes radii upon gel filtration. The calculated molecular weights of the two forms of alpha are 100 000 and 215 000; the frictional ratios are 1.34 and 1.65. This and other data suggest a possible monomer-dimer relation. In contrast, delta sediments uniformly at 6.5 S and also behaves uniformly upon gel filtration at 4595 A. The molecular weight of delta is distinct at 122 000; its frictional ratio is 1.39. Because of similarities of the DNA polymerizing activities of both forms of alpha and of delta, it is postulated that alpha is derived from delta by structural modification, resulting in a decrease in molecular weight, the tendency to aggregate as dimers, and a concomitant loss of 3' to 5' exonuclease activity.

Isolation of the catalytic core of DNA polymerase alpha from rabbit bone marrow

“…A representative purification of DNA polymerase alpha is outlined in Table 1. In comparison to our previously reported result (11) the yield of enzymatic activity has been increased by the inclusion of PMSF and PEG in the enzyme buffers. Previously, 7500 U of DNA polymerase were obtained from similar starting material after Step III; since these changes generally close to 40,000 U are obtained.…”

“…The pH and conductivity of solutions were measured with a Radiometer pH meter and a Yellowspring conductivity bridge. Purification of DNA Polymerase Alpha DNA polymerase was purified from erythroid hyperplastic rabbit bone marrow through Step VIII but with several important modifications of previously described procedures (11,17). Briefly, rabbits were injected with phenylhydrazine to induce erythroid hyperplasia.…”

Replication of Eukaryotic Chromosomes: A Close-up of the Replication Fork

“…Three forms of a-polymerase from HeLa cells are equally sensitive to aphidicolin, but one is significantly more sensitive to butylanilinouracil (59). A form of a-polymerase purifi ed from yeast (101, 102) and mammalian cells (103,104,462) has an associated 3'-5' exonuclease activity similar to that of HSV-l (105) and vaccinia virus (106) DNA polymerase. A mammalian enzyme from rabbit bone marrow, S-polymerase, may be the same as the D form of a-polymerase from calf thymus (95); both prefer poly d(A-T) as a primer template.…”

Thymidine Uptake, Incorporation and Dna Polymerase Activity in Murine Bladder Tumor Cell,mbt–2, Exposed to Uv Activated Dihematoporphyrin Ether

“…Data on effects of the photoactivated DHE on DNA polymerase a and p in a permeabilized cell system indicated a strong inhibition in the activity of DNA polymerase a and a mild stimulation of the DNA polymerase p. Current concepts on the function of DNA polymerases in mammalian cells suggest that polymerases (Y and S are largely responsible for DNA replication in nuclei (Hammond et al, 1987;Weissback, 1979). DNA polymerase p has been implicated in DNA repair synthesis; however, recent studies also indicated an involvement of DNA polymerase a in DNA repair process (Berger er al., 1979;Byrnes and Black, 1985;Chung et a/., 1983;Thommes et al, 1986). In R3230 AC mammary tumor, Crute et al (1986) Fig.…”