The 21 S complex has been purified over 2,000-fold and the partially purified enzyme complex contains in addition to SV40 DNA in vitro replication activity; a 640 kDa multiprotein DNA polymerase alpha-primase complex, a dA/dT sequence binding origin recognition protein, DNA-dependent ATPase, DNA ligase I, histone H1 preferring protein kinase, proliferating cell nuclear antigen (PCNA), topoisomerase I and RNase H.
The specific aims of the proposed project are the following. (1) To extensively purify the 21 S enzyme using conventional purification procedures, partially disassemble the complex by chromatography through coupled columns of native and denatured DNA-celluloses followed by anion exchange chromatography and purify the separated components. Reconstitution of the enzyme complex from its resolved components will be attempted. The steps of initiation, leading and lagging strand synthesis and supercoiling during T-antigen dependent replication of SV40 DNA in vitro will be investigated using the purified 21 S enzyme complex, reconstituted complex and purified subassemblies of the enzyme complex. (2) To determine the level of activity and organization of the 21 S complex of enzymes for DNA synthesis during the transition from G, to S and S to G2 phases of the cell cycle in synchronized HeLa cells in culture. (3) To investigate by immunofluorescent imaging analysis, using a variety of antibodies to components of the 21 S enzyme complex, the intracellular localization of the enzyme. The overall aim of the proposed project is to define possible controls for DNA biosynthesis during normal and abnormal growth. The immediate goal is to define the organization and control of the enzymatic machinery for chromosomal DNA replication in human cells. Our approach to this objective is to use simian virus 40 (SV40) DNA as a model replicon to study the human cell enzymatic machinery that participates in the initiation of T-antigen dependent replication of plasmid DNAs containing inserts of SV40 DNA containing sequence of the origin for replication. This approach led to the isolation from HeLa cell extracts of a 21 S megadalton complex of enzymes for DNA synthesis. The enzyme complex contains all of the activity for T-antigen-dependent and SV40 origin specific initiation of SV40 DNA replication in vitro.
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