Genome maintenance in hyperthermophilic Archaea

The discovery of hyperthermophilic microorganisms in thermal and hydrothermal vents has repelled the limits of life on the earth. Hyperthermophilic Archaea not only resist to extreme physico-chemical conditions like hot temperatures, high pressures, reduced fluids, but are perfectly adapted to these harsh environments. This implies original molecular and cellular mechanisms since cellular functions have to be maintained in these conditions. Several research groups around the world have been interested in DNA replication mechanisms in hyperthermophilic Archaea, first because of potentially biotechnological applications and also to understand how genomic maintenance can occur under these extreme environments. On the other hand, genomic comparisons between Archaea and Eukaryotes have shown homologous proteins and conserved mechanisms in these two groups. Therefore Archaea can be considered as a simplified model of Eukaryotes and also can help understanding how eukaryotic DNA replication has originated during evolution.

Part of the UMR 6197 laboratory is working on these problems.

In order to replicate their genome, all living species have enzymes which use single-stranded DNA as a template coupled to a oligonucleotide primer with a free 3’-OH terminus: the DNA polymerases.

These enzymes belong to six families depending on their protein sequenne : A, B, C, D, X and Y, and each of these families comprises several DNA polymerases displaying various cellular functions. Also DNA polymerases regulate their activities and implication in genomic maintenance by interacting with various cellular protein partners.

DNA replication can be divided into three steps: initiation, elongation and Okazaki fragments maturation.

The research projects of the group are centered on the functional characterization of the Pyrococcus abyssi replication complex:

- characterization of DNA polymerases ; identification of the replicase(s) acting on leading and lagging strands; properties of DNA polymerases B and D on damaged and undamaged DNA; properties of the DNA primase ; analysis of PCNA-binding motifs;
- characterization of replication factors, particularly the PCNA-loading by the RF-C ;
- Physical and functional interactions between partners of the P. abyssi DNA replication complex.
- Search for novel interacting partners of the P. abyssi DNA replication complex.

These are the research projects for the next quadriennal contract:

Translesion properties of Pyrococcus abyssi DNA polymerases B and D.

Results describe translesional properties of the PabPols in presence of AP sites that are dependent upon:
- reaction conditions (enzyme concentration),
- DNA substrate configuration.
Tolerance to AP sites differs among wild-type and exo- versions of the PabPols. Other types of DNA lesions are under investigation, particularly bulky adducts such as thymine dimers. These studies will also give insight into the flexibility of the active sites of the two DNA polymerases. Depending on the results, it should be important to obtain 3D structures of DNA polymerases in presence damaged and undamaged DNA templates.

Structural and functional characterization of Pyrococcus abyssi DNA polymerases.

Research will be made on the function of DNA polymerases at the Pyrococcus abyssi replication fork:
- What is the minimal DNA fragment synthesized to induce switching between PabPol D and PabPol B ? The experimentations will be made using fluorescently labelled DNA and mutated DNA polymerases (for polymerization and 3’-5’ exonuclease activities).
- What minimal size of a RNA fragment is needed for the activity and binding of PabPol B ? This is to know if DNA polymerase B is involved only in leading strand synthesis or also could participate in lagging strand synthesis.
- 3 D structure resolution of Pyrococcus abyssi DNA polymerases. After a first attempt on PabPol D in the frame of the REPBIOTECH program, a new approach is undertaken in collaboration with the laboratory of Prof. Delarue, at the Pasteur Institute, also on PabPol B.

PCNA : a platform for genomic maintenance

The aim of the program is to identify amino acids involved in PCNA interactions with the protein partners and DNA, as well as the conformational changes at the inframolecular level. Technical facilities for the project are available in the laboratory as well as at the Brest university (laser YAG). Also, SAXS and SANS experimentations will be performed in collaboration. In fine, the results should give insight into conformational changes of PCNA, at the global and local levels, in a cellular context (particularly quantitative data which can not be obtained by other techniques like crystallography and micropscopy), and identifying essential amino acids pertaining to the function of PCNA.

Characterization of novel interacting partners of the Pyrococcus abyssi DNA replication complex as related to genomic maintenance.

Recently, the LM2E has engaged, particularly in the frame of the REPBIOTECH, Marine Génomique (ANR) et Marine Genomics Europe (Framework of Excellence) programs, in the serach for new interacting partners of DNA replication proteins. This work is performed in collaboration with the group of H. Myllykallio (université de Paris-Sud). In order to obtain more cellular baits, the laboratory is partner of the Marine-Express program (REX Marine Genomics Europe) consisting in a medium scale protein surexpression platform. The project should allow us to obtain a high number of recombinant proteins involved in DNA metabolism (144 potential targets over 3 years), therefore giving a global insight into archaeal genomic maintenance at the level of the organism.

Photo with the kind permission of Rudolf Ladenstein (Institut Karolinska, Sweden) and Jean-Paul Raffin (CNRS, F)