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[RNA and DNA synthesis catalyzed by a DNA-polymerase alpha complex with primase from silkworm cells on
Abstract:
Depending on the ionic environment the replicative complex of silkworm Bombyx mori, containing DNA polymerase alpha and primase, catalyzes on single-stranded DNA of phage M13 a NTP-dependent synthesis or elongation of preformed primers. In the presence of NTPs and dNTPs at conditions optimal for the NTP-dependent synthesis the replicative complex synthesizes on M13 DNA oligoribonucleotides of 9-11 residues, which serve as primers for polymerization of DNA. The length of RNA-primers synthesized by primase of the complex depends on concentration of dNTP but does not depend on activity of DNA polymerase alpha. During elongation of exogenic primers annealed to M13 DNA the complex is processive synthesizing DNA fragments of dozens residues without dissociation from the template. Double-stranded structures in DNA such as "hairpins" appear to be barriers for driving of the complex along the template and cause pauses in elongation. DNA-binding proteins the SSB of Escherichia coli or the p32 of phage T4 destabilize double-stranded regions in DNA and eliminate elongation pauses corresponding to these regions. The replicative complex is able to fill in single-stranded gaps in DNA completely and to perform slowly the synthesis with displacement of one of parent strands in duplexes via repeated cycles of binding to the primer-template, limited elongation and dissociation.
Insights
The silkworm replicative complex synthesizes RNA primers and elongates DNA, with its processivity affected by DNA structures and binding proteins. This complex can fill DNA gaps and perform strand displacement synthesis.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- The replicative complex from silkworm Bombyx mori contains DNA polymerase alpha and primase.
- This complex functions in DNA replication, utilizing nucleotide synthesis and primer elongation.
Purpose of the Study:
- To investigate the catalytic activities of the Bombyx mori replicative complex on single-stranded phage M13 DNA.
- To understand the factors influencing primer synthesis and DNA elongation, including ionic environment and DNA secondary structures.
Main Methods:
- Enzymatic assays using phage M13 DNA as a template.
- Analysis of DNA polymerase alpha and primase activities under varying conditions.
- Investigating the effect of DNA-binding proteins (E. coli SSB, phage T4 p32) on complex function.
Main Results:
- The complex catalyzes NTP-dependent synthesis of RNA primers (9-11 residues) and elongation of preformed primers.
- Primer length depends on dNTP concentration; elongation is processive but paused by DNA hairpins.
- DNA-binding proteins destabilize double-stranded DNA, resolving elongation pauses.
- The complex can fill single-stranded gaps and perform slow strand displacement synthesis.
Conclusions:
- The Bombyx mori replicative complex exhibits versatile DNA synthesis capabilities, including primer synthesis, processive elongation, gap filling, and strand displacement.
- DNA secondary structures and accessory proteins significantly modulate the complex's processivity and function during DNA replication.