DNA SYNTHESIS: REGULATION IN NORMAL AND CANCER CELLS

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The objective of this research is to define possible controls for DNA biosynthesis during normal and abnormal growth. A multiprotein form of DNA polymerase (polymerase alpha2) has been purified to homogeneity from the combined low-salt extract (0.15M KCl) of nuclei and cytoplasm of HeLa cells. DNA polymerase alpha2 has been resolved into its catalytic and subunit structure by hydrophobic affinity and ion exchange chromatography and chromatofocusing. It is composed of a DNA polymerase alpha catalytic core enzyme (Mr = 180,000), a "primase" (Mr = 70,000), 5',5''' diadenosine tetraphosphate (Ap4A) binding protein (Mr = 92,000, a dimer of 47,000), a single-strand specific exonuclease (Mr = 69,000) that has a proof-reading function in vitro and two accessory proteins Cl (Mr = 96,000, a tetramer of 24,000) and C2 (Mr = 52,000) that function as primer-recognition proteins in vitro with primed single-stranded DNA templates. Polyclonal antibodies (rabbit) have been prepared against the homogeneous DNA polymerase alpha catalytic core subunit and the Ap4A binding protein. The antibodies are being used to study the topology of the interactions of the multiprotein subunits of DNA polymerase alpha2. The antibodies to polymerase alpha and Ap4A are being used to study the interactions of DNA polymerase alpha2 and Ap4A in chromosome replication. Western blot analysis of erude HeLa cell homogenates and purified DNA polymerase alpha2 with the antibody to the polymerase alpha catalytic core subunit shows a single band of Mr 183,000. All of the DNA polymerase alpha2 is extractable from nuclei by 0.15M KCL. The association of DNA polymerase alpha core subunit and C1, C2 proteins with the primase appear to regulate the size of oligoribonucleotide primer that is synthesized. DNA polymerase alpha-primase complex synthesizes RNA primers of 7 to 15 ribonucleotides in legnth on defined DNA templates. The DNA polymerase alpha2 associated primase synthesizes pentanucleotide primers on the same defined DNA templates. We are studying the interactions of DNA polymerase alpha2 and other components of the DNA synthesizing machinery in the in vitro replication of the SV40 DNA. (I)