In vitro reconstitution of RNA primer removal in Archaea reveals the existence of two pathways

Type Article
Date 2012-10
Language English
Author(s) Henneke GhislaineORCID1, 2, 3
Affiliation(s) 1 : IFREMER, UMR 6197, Lab Microbiol Environm Extremes, F-29280 Plouzane, France.
2 : Univ Bretagne Occidentale, UMR 6197, Lab Microbiol Environm Extremes, F-29280 Plouzane, France.
3 : Ctr Natl Rech Sci, UMR 6197, Lab Microbiol Environm Extremes, F-29280 Plouzane, France.
Source Biochemical Journal (0264-6021) (Portland Press Ltd), 2012-10 , Vol. 447 , P. 271-280
DOI 10.1042/BJ20120959
WOS© Times Cited 22
Keyword(s) Archaea, DNA ligase, DNA polymerase, nuclease, Okazaki fragment
Abstract 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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