ISOLATION AND CHARACTERIZATION OF DNA POLYMERASE-EPSILON FROM THE SILK GLANDS OF BOMBYX-MORI

The silk gland of Bombyx mori, an endomitotically replicative tissue s hows high levels of DNA polymerases alpha, delta, and epsilon activiti es. The ratio of polymerase alpha to that of delta plus epsilon is mai ntained at 1.1 to 1.3 in both the posterior and middle silk glands for the entire duration of late larval development. The three activities copurify in the initial stages of fractionation through phosphocellulo se and DE52 but polymerase alpha gets resolved from the others on hydr oxylapatite column. Separation between polymerase delta and epsilon is achieved by chromatography on QAE-Sephadex. DNA polymerase epsilon is a heterodimer comprising of 215- and 42-kDa subunits. The activity is maximum at pH 6.5 and the K(m) values for dNTPs vary between 3-9 muM. The enzyme possesses an intrinsically associated exonuclease activity which functions in the mismatch repair during DNA synthesis. Both pol ymerase and 3' --> 5' exonuclease activities are associated with the 2 15-kDa subunit. By itself, DNA polymerase epsilon is processive and th e catalytic activity is not enhanced by externally added bPCNA (Bombyx -proliferating cell nuclear antigen, an auxiliary protein for DNA poly merase delta). The enzyme resembles polymerase delta in having the exo nuclease activity and in its response to aphidicolin or substrate anal ogs, but could be distinguished from the latter by its lack of respons e to the bPCNA and sensitivity to dimethyl sulfoxide. The two enzymes show partial immunological cross-reactivity with each other but no imm unological relatedness to polymerase alpha. The absence of the repair enzyme DNA polymerase beta and the presence of substantial levels of p olymerase epsilon in the silk glands suggest a possible role for the l atter in DNA repair in that tissue.