Paradoxes of in Situ Polyacrylamide Gel Assays for DNA Polymerase Priming
The DNA polymerase-α from Drosophila melanogaster embryos is now one of the most extensively characterised eukaryotic multisubunit polymerases because it can be isolated in high yields in an intact, yet highly purified form. It has proved to be amenable to detailed biochemical studies of its subunit structure and subunit contributions to the overall DNA synthesis reaction, primer and template requirements, processivity, cellular functions and immunological properties (1–10). The holoenzyme utilises not only gapped duplex DNA as a primer-template system, but also pre-DNA and pre-RNA primed single-stranded circular phage DNAs (6, 10). Furthermore, it will synthesise DNA on an unpreprimed single DNA strand in the presence of both rNTPs and dNTPs because it also catalyses the synthesis of short RNA and RNA-DNA oligonucleotides which prime subsequent DNA chain elongation (8, 9). Whether this composite reaction is related to Okazaki fragment initiation and synthesis in vivo remains to be determined.
KeywordsGlycerol Gradient Template Requirement Fragment Initiation Test Tube Assay Page Assay
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