Abstract

In Archaea, the proteins involved in the genetic information processing pathways, including DNA replication, transcription, and translation, share strong similarities with those of eukaryotes. Characterizations of components of the eukaryotic-type replication machinery complex provided many interesting insights into DNA replication in both domains. In contrast, DNA repair processes of hyperthermophilic archaea are less well understood and very little is known about the intertwining between DNA synthesis, repair and recombination pathways. The development of genetic system in hyperthermophilic archaea is still at a modest stage hampering the use of complementary approaches of reverse genetics and biochemistry to elucidate the function of new candidate DNA repair gene. To gain insights into genomic maintenance processes in hyperthermophilic archaea, a protein-interaction network centred on informational processes of Pyrococcus abyssi was generated by affinity purification coupled with mass spectrometry. The network consists of 132 interactions linking 87 proteins. These interactions give insights into the connections of DNA replication with recombination and repair, leading to the discovery of new archaeal components and of associations between eucaryotic homologs. Although this approach did not allow us to clearly delineate new DNA pathways, it provided numerous clues towards the function of new molecular complexes with the potential to better understand genomic maintenance processes in hyperthermophilic archaea. Among others, we found new potential partners of the replication clamp and demonstrated that the single strand DNA binding protein, Replication Protein A, enhances the transcription rate, in vitro, of RNA polymerase. This interaction map provides a valuable tool to explore new aspects of genome integrity in Archaea and also potentially in Eucaryotes.

Highlights

  • The control of genome integrity is of pivotal importance for the cell

  • The proteins identified by mass spectrometry, that were present in both AP/MS experiments and for which the Mascot score was more than 5 fold higher in the assays compared to the negative controls, were regarded as being able to interact with the bait under consideration

  • We reasoned that this exploratory analysis should unravel new and unsuspected relationships between known factors and novel components involved in nucleic acid processes, as well as potential moonlighting proteins linking DNA maintenance with distinct metabolic pathways and we only used the objective criterion of significance ratio (SR) score to classify the proteins identified in mass spectrometry, rather than excluding proteins based on functional annotations

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Summary

Introduction

The control of genome integrity is of pivotal importance for the cell. Failure to maintain genome stability results in the accumulation of mutations, genome rearrangements and cell death. Molecular interactions involved in DNA repair and recombination and their coordination with DNA synthesis are required for genome maintenance These genome transactions and their proper coordination require the assembly of multiprotein machines at specific sites at the chromosome or chromatin level. The wide variety of lesions that can arise in DNA and the biological importance of coping with these are reflected by the diversity of biochemical strategies for DNA repair found in model micro-organisms. In this context, Hyperthermophilic Archaea (HA) are of special interest as DNA in hyperthermophiles is exposed to temperatures that increase the rate of decomposition [1]. In the hyperthermophilic euryarchaeon P. abyssi, the level of apurinic/

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