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A cell-free system for the replication fo bacteriophage M-13 duplex DNA
Nucleic Acids Research
|May 1, 1978
Summary
This study shows that cell-free extracts from M-13 infected E. coli can replicate M-13 DNA. Specific proteins and inhibitors affect the synthesis of viral and complementary DNA strands.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- M-13 phage is a single-stranded DNA virus that infects Escherichia coli.
- Understanding M-13 DNA replication is crucial for studying viral replication mechanisms.
Purpose of the Study:
- To investigate the cell-free replication of endogenous M-13 duplex DNA using concentrated extracts.
- To identify factors influencing the synthesis of viral and complementary DNA strands.
Main Methods:
- Utilized cell-free extracts from M-13 am5 infected E. coli concentrated on cellophane disks.
- Employed bromodeoxyuridine triphosphate for labeling newly synthesized DNA.
- Assessed the effects of ATP, inhibitors (nalidixic acid, novobiocin, arabinosylnucleoside triphosphates, rifampicin), and helix-destabilizing proteins (M-13 gene V, Eco HD-protein).
Main Results:
- Efficient replication of M-13 duplex DNA was observed in cell-free extracts.
- Newly synthesized DNA predominantly formed hybrid molecules (viral or complementary strand labeled) and some fully synthetic duplex DNA.
- DNA synthesis required ATP and was inhibited by specific agents.
- Rifampicin and M-13 gene V protein preferentially inhibited complementary strand synthesis.
- Eco HD-protein stimulated synthesis of both hybrid types and fully synthetic DNA.
Conclusions:
- Cell-free M-13 DNA replication is feasible and influenced by various factors.
- Specific helix-destabilizing proteins play distinct roles in modulating viral DNA strand synthesis.