[Genetic and biochemical studies on the mechanism of mammalian chromosome replication]

It has been clarified that basic mechanism of deoxyribonucleic acid (DNA) replication is conserved from bacteria to higher cells. What distinguishes prokaryotic and eukaryotic modes of DNA replication most clearly is that bacterial chromosomes form a single replicon copied from a single initiation point and eukaryotic chromosomes consist of multiple replicons that initiate at multiple points. Thus, eukaryotes have to coordinate orderly replication of the genome. In order to understand this complex problem as a whole, three approaches were chosen. First approach is a genetic one. Certain number of temperature-sensitive (ts) mutants were isolated from mouse FM3A cells. One of the ts mutants, designated as tsFT20, was shown to contain heat-labile DNA polymerase alpha (pol. alpha). By the use of this mutant strain, it was proved that pol. alpha is essential for mammalian DNA replication. In addition, the human gene for pol. alpha on the X chromosome was assigned. Second approach is an enzymological one. FM3A cells were used for the identification and characterization of enzymes and proteins supposed to be involved in DNA replication. Four DNA-dependent ATPases, three pol. alpha stimulation factors, DNA topoisomerases I and II have been identified, as well as a stimulation factor for the assembly of nucleosome. DNA helicase activity was detected in two of the DNA-dependent ATPase (B and C1). Third approach is the reconstitution of DNA replication in cell-free system. By use of polyoma virus DNA as a template, cell-free extract from FM3A cells supported DNA replication in the presence of polyoma virus large T-antigen. This cell-free system will be useful for the analysis of the function of replication enzymes and proteins as well as the characterization of ts mutants.