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A DNA primase activity associated with DNA polymerase alpha from Drosophila melanogaster embryos
Abstract:
Preparations of DNA polymerase alpha from early embryos of Drosophila melanogaster catalyze the ATP-dependent synthesis of DNA with single-stranded M13 DNA or poly(dT) templates. In the case of M13 DNA, GTP, but not UTP or CTP, can replace ATP. The reaction is completely dependent on added template and is not inhibited by alpha-amanitin. Alkaline hydrolysis of the product synthesized in the presence of [alpha-32P]dATP and poly(dT) generates 32P-labeled 3'(2') adenylate, showing that a covalent ribo-deoxynucleotide linkage is formed. Furthermore, incorporation of ribonucleotides occurs at the 5' end of the newly synthesized polynucleotide chain. These findings are consistent with the hypothesis that a ribo-oligonucleotide primer is synthesized by primase action and subsequently elongated by DNA polymerase. Under the appropriate conditions, DNA polymerase I from Escherichia coli can elongate primers formed by primase in the presence of ATP and poly(dT). Primase activity copurifies with DNA polymerase alpha and may be part of the multisubunit polymerase molecule.
Insights
Drosophila DNA polymerase alpha synthesizes DNA using ribonucleotide primers, a process essential for DNA replication. This primase activity may be an integral part of the DNA polymerase alpha enzyme complex.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA replication requires DNA polymerases to synthesize new DNA strands.
- The initiation of DNA synthesis often involves RNA primers.
Purpose of the Study:
- To investigate the mechanism of DNA synthesis catalyzed by DNA polymerase alpha from Drosophila melanogaster.
- To determine the role of ribonucleotides in DNA synthesis initiation.
Main Methods:
- Enzymatic assays using single-stranded M13 DNA and poly(dT) templates.
- ATP-dependent DNA synthesis.
- Alkaline hydrolysis of synthesized DNA products.
- Analysis of ribonucleotide incorporation.
Main Results:
- DNA polymerase alpha catalyzes ATP-dependent DNA synthesis.
- Ribonucleotides are incorporated at the 5' end, forming a ribo-deoxynucleotide linkage.
- Primase activity, responsible for primer synthesis, copurifies with DNA polymerase alpha.
Conclusions:
- DNA polymerase alpha likely synthesizes a ribo-oligonucleotide primer.
- This primer is subsequently elongated by DNA polymerase activity.
- Primase activity may be an intrinsic function of the DNA polymerase alpha complex.