DNA polymerase α-DNA primase from human lymphoblasts

Abstract

The DNA polymerase α-DNA primase complex from the human lymphoblast line HSC93 has been enriched to near homogeneity by using an immunoaffinity purification protocol which was developed earlier for the purification of the calf thymus enzyme (Nasheuer, H.-P. and Grosse, F. (1987) Biochemistry 26, 8458–8466). Immunoaffinity purified polymerase-primase from human cells consisted of four subunits displaying molecular weights of 195000 and 180000 for the DNA synthesizing α-subunit, of 68000 for the β-subunit, and of 55000 and 48000 for the primase-carrying γ- and δ-subunit, respectively. The isoelectric pH values for the individual subunits were estimated from non-equilibrium pH gradients to be between 5.9 and 5.7 for the α-subunit, at 5.5 for the β-subunit, and at 7.5 and 8.0 for the γ- and δ-subunit, respectively. The purified polymerase-primase converted single-stranded ΦX174 DNA into the double-stranded form in a primase-initiated reaction. During this process, 3–10 RNA primers were formed. RNA primers were about 11 nucleotides long. Elongation of existing RNA primers by the human polymerase-primase was semi-processive; following primer binding the DNA polymerase continuously incorporated 20 to 50 nucleotides, then it dissociated from the template DNA.