Kinetic characteristics which distinguish two forms of calf thymus DNA polymerase .alpha.

Abstract

DNA polymerase .alpha. was isolated to yield 5 nuclease-free forms of .alpha.-polymerase, A1, A2, B, C and D. Holmes et. al. have suggested that the C form is the core enzyme of .alpha.-polymerase and have demonstrated that removal of a protein subunit from the A1 form yields an enzyme with the physical properties of the C form. They did not investigate the function of the subunit because the A1 and C forms were not easily distinguished with biochemical kinetics. The present study demonstrates 3 kinetic differences between these forms: the .alpha.-A1-polymerase adds more nucleotides per binding event to activated DNA (is more processive) than does .alpha.-C-polymerase; the synthetic activity of the .alpha.-A1-polymerase is greater on a template with an average gap size of 65 nucleotides than it is on a template with an average gap size of 10 nucleotides whereas that of the .alpha.-C-polymerase is not; and the synthetic activity of the .alpha.-C-polymerase is inhibited by high concentration of activated calf thymus DNA (> 300 .mu.M) whereas that of the .alpha.-A1-polymerase is not. The nature of the inhibitor was investigated and found to be a nuclear RNA component present in the DNA preparations. These kinetic differences may provide a means to assay for the protein subunit that converts .alpha.-C-polymerase to .alpha.-A1-polymerase, and provide a basis for isolation and characterization of other DNA replication-assciation proteins.