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Okazaki fragment maturation mechanisms

Reconstitution of Okazaki fragment maturation in P. abyssi.

Prior to the start of REPBIOTECH project, we had detected in P. abyssi short nascent DNA strands with 5' RNA segments whose size (up to 120 base pairs) and structure are similar to eukaryotic Okazaki fragments, indicating that Archaea use RNA primers synthesised by eukaryotic-like primase for replication initiation of leading strands and for synthesis of all Okazaki fragments at the retrograde arm of the replication fork (Myllykallio and Forterre 2000; Myllykallio et al. 2000). The details of how these primers are synthetized and removed during chromosomal DNA replication in Archaea are poorly understood.

The apparent absence of DNA polymerase alpha (required for low fidelity synthesis of DNA primers of eukaryotic Okazaki fragments) had suggested that synthesis of Okazaki fragments is likely to possess unique features in Archaea. At the start of the project, proteins homologous to eukaryotic Dna2 helicase/endonuclease that is required for removal of long RNA/DNA primers from eukaryotic Okazaki fragments had been detected in Archaea. However, during the early phases of our project, biochemical work demonstrated that archaeal Dna2-like proteins are inhibited by 5 RNA segments, strongly suggesting that they are not orthologous to their eukaryotic homologs. Consequently, we have not pursued further the role of archaeal Dna2-like proteins in this project.

To address how RNA primers are removed from archaeal Okazaki fragments, we have cloned and overexpressed a number of P. abyssi proteins with an expected function in maturation of Okazaki fragments (e.g. PCNA replication clamp, Fen1 nuclease, DNA ligase). Our interaction studies using surface plasmon resonance revealed high-affinity interactions between PCNA, Fen1 and DNA ligase I as well as RNaseHII.

We have also identified a novel physical interactions between Fen1-RNase HII, suggesting that activities of RNase HII and Fen1 could be coupled by PCNA during DNA replication and/or repair. Physiological significance of the above interactions detected in vitro were confirmed using pull-down assays. Moreover, physical interactions increased also enzymatic activities of the both Fen1 nuclease and DNA ligase. Altogether these results led to functional model for the Okazaki fragment maturation in Archaea.

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Ecole Polytechnique