Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo
A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase alpha, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4/sup 0/C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases.
- Research Organization:
- Dept. of Pathology, Univ. of Vermont College of Medicine, Burlington, VT (US)
- OSTI ID:
- 7027479
- Journal Information:
- Mol. Cell. Biol.; (United States), Vol. 8:5
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
CELL CYCLE
CHROMOSOMES
STRUCTURE-ACTIVITY RELATIONSHIPS
DNA POLYMERASES
BIOCHEMICAL REACTION KINETICS
DNA REPLICATION
BIOLOGICAL PATHWAYS
MOLECULAR BIOLOGY
MITOCHONDRIA
BIOSYNTHESIS
CELL FLOW SYSTEMS
CYTOPLASM
EXPERIMENTAL DATA
FOLIC ACID
GENE AMPLIFICATION
IN VITRO
IN VIVO
OXIDOREDUCTASES
SPECIFICITY
TECHNOLOGY ASSESSMENT
AMINO ACIDS
AROMATICS
AZAARENES
CARBOXYLIC ACIDS
CELL CONSTITUENTS
DATA
DRUGS
ENZYMES
HEMATINICS
HEMATOLOGIC AGENTS
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
INFORMATION
KINETICS
NUCLEIC ACID REPLICATION
NUCLEOTIDYLTRANSFERASES
NUMERICAL DATA
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
PTERIDINES
REACTION KINETICS
SYNTHESIS
TRANSFERASES
VITAMIN B GROUP
VITAMINS
550200* - Biochemistry
550300 - Cytology
550400 - Genetics