Isolation and characterisation of thermostable DNA polymerases in the presence of DNA lesions in the hyperthermophilic archaeon, P. abyssi. Examination of conventional and non conventional mechanisms in the genome maintenance of microorganisms inhabiting extreme environments.
ARCHPOL intends to examine the spectra of damaged nucleic acids in P. abyssi, under normal and stressful physiological conditions. Identification and quantitative analyses target the genome and the precursors (dNTPs and NTPs). ARCHPOL also aims at catching novel DNA polymerising enzymes inducible by genotoxic stresses. This goal is sustained by the lack of specialised DNA polymerases (translesional DNA pol) and the limited number of conventional DNA polymerases in the genome of P. abyssi. Biochemical studies of specialised and conventional DNA polymerases (families B and D) in the presence of damaged nucleic acids will be detailed. Thus, ARCHPOL gains more insight into the tolerance of damaged nucleic acids in unusual microoganisms via the concept of functional diversity of thermostable DNA polymerases. In addition to fundamental questions raised by this topic, there are also biotechnological objectives. Finding novel thermostable DNA polymerases able to PCR amplify damaged DNA matches the present demand of industrials. These novel marketed enzymes could find applications in forensics or paleogenetics.
A gas-lift bioreactor is used to cultivate P. abyssi under normal physicochemical conditions and to simulate environmental fluctuations occurring at hydrothermal vents.
Collected cell extracts are utilised to identify damaged nucleic acids and to isolate novel DNA polymerising activities. The detection of damages is performed by HPLC-UV coupled to electrochemistry or mass spectrometry. Isolation of DNA polymerases is based on immunodepletion of cell extracts from replicative DNA polymerases (polB and polD) followed by affinity purification and activity gels.
Functional and structural characterisation of family B DNA polymerase from P. abyssi indicated that the uracil binding pocket (uracil, product arising from spontaneous deamination of cytosine and accelerated at elevated temperature) can also accommodate the four canonical bases from genomic DNA. A base-checking role of the template is proposed. This phenomenon seems to be unique in Archaea.
Functional characterisation of family D DNA polymerase from P. abyssi in the presence of uracil has shown an inhibitory effect on DNA polymerising activity, reducing the rate of DNA synthesis. On the other hand, uracil stimulated the degradation of DNA primers annealed to DNA template. A molecular mechanism of uracil recognition different to that of DNA polymerase B is proposed.
The optimisation of activity gels for detecting novel DNA polymerising activities has been challenged. Instead of radioactivity, fluorescent probes labelling the polymerised DNA fragments have been used. The labelling method relied on different chemistries for cross-linking the probes to the DNA fragments. This new methodology is not optimised yet and is still under investigation. On the other hand, aberrant electrophoretic migration of degradation products released by exonuclease activities by DNA polymerases has been studied. This unanticipated work highlighted the importance of choosing the proper fluorescent dyes for electrophoresis. Indeed, we showed that a positively charged fluorescent dye can interfere with the migration profile of short DNA fragments (< 8 nt in length).
DNA polymerase D from P. abyssi exhibited PCR performance higher than Taq, the enzyme routinely used in research laboratories. In particular, polD seems to tolerate common PCR inhibitors present in human samples. These properties could be very useful in forensics and paleogenomics, for instance.
The inhibitory effect of uracil on DNA polymerase activities by conventional DNA polymerases (families B et D) raises the question of the occurrence of uracil in genomic DNA and in the nucleotides pool in Archaea. The identification and quantification of this damage has not been assessed by ARCHPOL but will be determined in the future. This task is very important to be addressed not only in Archaea but also in Bacteria and eukaryotes. On the other hand, the impact of the oxidative 8-oxodG DNA lesion on polymerising activities by conventional DNA polymerases is under investigation. This DNA lesion is present in the genome of P. abyssi and is more abundant than in the genome of the bacteria, Escherichia coli.
A functional and structural study showing the capture of canonical bases in the uracil recognition pocket of family B DNA polymerase has been published in August, 2012. Jérôme Gouge, Céline Ralec, Ghislaine Henneke and Marc Delarue. Molecular Recognition of Canonical and Deaminated Bases by P. abyssi Family B DNA Polymerase. J. Mol. Biol. (2012) 423, 315–336.
The inhibition of DNA polymerising activity and the stimulation of DNA primer degradation by family D DNA polymerase gave rise to the following paper; Richardson TT, Gilroy L, Ishino Y, Connolly BA, Henneke G. Novel inhibition of archaeal family-D DNA polymerase by uracil. Nucleic Acid Research (2013) 41, 4207-18
The article about aberrant electrophoretic migration of positively charged fluorescent dyes of short DNA fragments has been published in the electrophoresis journal in 2014: Anomalous electrophoretic migration of short oligodeoxynucleotides labelled with 5’-terminal Cy5 dyes. Tom Killelea, Christine Saint-Pierre, Céline Ralec, Didier Gasparutto, Ghislaine Henneke.
The objective of ARCHPOL is to discover novel functional DNA polymerases in hyperthermophilic archaea and to examine the potential impact of relevant damaged nucleic acids onto their intrinsic properties. This interest is sustained by the limited number of DNA polymerases identified to date in the genome of the hyperthermophilic model, P. abyssi, posing the intriguing question of how this microorganism evolve to deal with a specific threshold of DNA damage without affecting cell growth and viability. Despite efficient DNA repair strategies employed to remove damaged nucleic acids, some of them persist. Palud. A et al recently published that DNA polymerases from P. abyssi develop unique and efficient features to counteract residual DNA lesions. In ARCHPOL, the functional analyses of conventional and damaged-induced DNA polymerases will be detailed in the presence of relevant damaged nucleic acids. This project aims at giving a clear picture of the involvement of DNA polymerases in damage tolerance in hyperthermophilic archaea that may provide useful information to further refined model of genomic maintenance in all living organisms.
Unique and innovative methods will be applied to discover novel DNA polymerases and to identify the types and occurrence of damaged nucleic acids. A gas-lift bioreactor which offers the possibility to inflict genotoxic stresses will be used to produce the P. abyssi biomass. The types and rates of damaged nucleic acids will be assessed by cutting-edge analytic methods. Identification and captured of novel DNA polymerases will be obtained by implemented and original techniques. When required, relevant DNA damage will be introduced into DNA template and functional characterization of appropriate DNA polymerases will be investigated.
As a consequence from basic fundamental research, ARCHPOL plans to develop valuable biochemical properties from P. abyssi polymerases that could be applied to various biotechnological applications. The current tendency in Polymerase chain Reaction (PCR) technology is to discover thermostable polymerases with broadened substrate spectra that could find applications in forensics and paleogenetics. In order to optimize the chances of success of ARCHPOL, which combines scientific and technological objectives, a highly competent and complementary team will be unified.
Madame Ghislaine Henneke (INSTITUT FRANCAIS DE RECHERCHE POUR L'EXPLOITATION DE LA MER (IFREMER)) – firstname.lastname@example.org
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
IFREMER INSTITUT FRANCAIS DE RECHERCHE POUR L'EXPLOITATION DE LA MER (IFREMER)
Help of the ANR 280,200 euros
Beginning and duration of the scientific project: - 36 Months