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Highly efficient copying of single-stranded DNA by eukaryotic cell chromatin
Nucleic Acids Research
|July 1, 1978
Summary
Eukaryotic cell chromatin efficiently synthesizes DNA using single-stranded DNA as a template. This DNA synthesis capability, primarily driven by DNA polymerase alpha, is reduced in G1-arrested cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Hepatoma tissue culture (HTC) cells in S phase incorporate [3H]dTMP into DNA.
- Chromatin structure and its role in DNA replication are key areas of study.
Purpose of the Study:
- To investigate the efficiency of DNA synthesis by eukaryotic cell chromatin using various DNA templates.
- To identify the enzyme responsible for copying denatured DNA.
Main Methods:
- In vitro DNA synthesis assays using chromatin from S phase and G1-arrested HTC cells.
- Stimulation assays with single-stranded DNA, activated DNA, and synthetic polynucleotides (poly(dC), poly(dT)).
- Enzyme characterization based on sensitivity to sulfhydril group blocking and ionic requirements.
Main Results:
- Chromatin significantly enhances DNA synthesis with single-stranded and activated DNA templates (40-60 fold stimulation).
- Poly(dC) and poly(dT) act as effective templates, copied complementarily by chromatin.
- DNA synthesis efficiency is considerably reduced in chromatin from G1-arrested cells.
- DNA polymerase alpha is tentatively identified as the enzyme responsible for denatured DNA copying.
Conclusions:
- Eukaryotic cell chromatin exhibits high efficiency in copying single-stranded DNA.
- DNA polymerase alpha is likely the primary enzyme involved in this process.
- Cell cycle stage (S vs. G1) impacts chromatin's ability to copy exogenous DNA templates.