Using model DNA substrates and purified recombinant proteins from Pyrococcus abyssi, I have reconstituted the enzymatic reactions involved in RNA primer elimination in vitro. In my dual-labelled system, polymerase D performed efficient strand displacement DNA synthesis, generating 5′-RNA flaps which were subsequently released by Fen1, before ligation by Lig1. In this pathway, the initial cleavage event by RNase HII facilitated RNA primer removal of Okazaki fragments. In addition, I have shown that polymerase B was able to displace downstream DNA strands with a single ribonucleotide at the 5′-end, a product resulting from a single cut in the RNA initiator by RNase HII. After RNA elimination, the combined activities of strand displacement DNA synthesis by polymerase B and flap cleavage by Fen1 provided a nicked substrate for ligation by Lig1. The unique specificities of Okazaki fragment maturation enzymes and replicative DNA polymerases strongly support the existence of two pathways in the resolution of RNA fragments.
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Research Article|
September 26 2012
In vitro reconstitution of RNA primer removal in Archaea reveals the existence of two pathways
Ghislaine Henneke
Ghislaine Henneke
1
*Ifremer, UMR 6197, Laboratoire de Microbiologie des Environnements Extrêmes, BP 70, 29280 Plouzané, France
†Université de Bretagne Occidentale, UMR 6197, Laboratoire de Microbiologie des Environnements Extrêmes, 29280 Plouzané, France
‡Centre National de la Recherche Scientifique, UMR 6197, Laboratoire de Microbiologie des Environnements Extrêmes, 29280 Plouzané, France
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Publisher: Portland Press Ltd
Received:
June 12 2012
Revision Received:
July 20 2012
Accepted:
July 31 2012
Accepted Manuscript online:
July 31 2012
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2012 Biochemical Society
2012
Biochem J (2012) 447 (2): 271–280.
Article history
Received:
June 12 2012
Revision Received:
July 20 2012
Accepted:
July 31 2012
Accepted Manuscript online:
July 31 2012
Citation
Ghislaine Henneke; In vitro reconstitution of RNA primer removal in Archaea reveals the existence of two pathways. Biochem J 15 October 2012; 447 (2): 271–280. doi: https://doi.org/10.1042/BJ20120959
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