Protection of DNA against Thermodegradation by Salts at Temperatures Typical for Hyperthermophiles

Evelyne Marguet & Patrick Forterre



Poster displayed during the first meeting of the Cell Factories EU BIOTECH Project, held in Athens, Greece, April 19-21 1997.
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Protection of DNA against thermodegradation by salts at temperatures typical for hyperthermophiles

Evelyne Marguet and Patrick Forterre

Institut de génétique et Microbiologie, Université Paris-Sud, 91405 Orsay Cedex, France.
 DNA molecules are exposed at very high temperatures (80-110°C) for long period in 
hyperthermophiles and for short periods in biotechnological procedures involving thermophilic 
enzymes. These temperature stress can potentially induces various chemical modifications, the 
main one being depurination followed by cleavage of the nearby phosphodiester bond. Heat-
induced DNA damages should be repaired in hyperthermophiles, but it is not clear to what extent 
their repair systems have to be be efficient. On the other hand, DNA damages induced by high 
temperature in vitro cannot be repaired and might lead to various types of error cascades. 

 Most studies on DNA thermodegradation in vitro have been initially performed using linear 
double-stranded DNA at temperatures below the Tm (usually 70°C-80°C). We have recently 
initiated similar investigations at temperatures more typical for hyperthermophiles, i.e. from 90 to 
110°C, using supercoiled plasmids. Plasmids are much more resistant to thermodenaturation than 
linear DNA, since the topological links between the two strands cannot be eliminated, as long as 
the they are covalently closed. We have shown that a bacterial plasmid is indeed resistant to 
denaturation, at least up to 107°C; but is rapidly degraded at such temperature (Marguet and 
Forterre, 1994). Although hyperthermophiles possess an enzyme, reverse gyrase, which 
produces positively supercoiled DNA (for review, see Forterre et al., 1996), positively 
supercoiled plasmids are no more resistant to thermodegradation than negatively supercoiled 
ones. However, subsequent thermodegradation was reduced in the presence of physiological 
concentrations of either monovalent or divalent salts (Marguet and Forterre, 1994). 

 Protection of DNA against thermodegradation by salts could be relevant for 
hyperthermophilic life, since some hyperthermophiles exhibit very high intracellular salt 
concentrations. They can be also significant for DNA manipulation in vitro . Here, we report 
further experiments on this problem. In particular, we have compared the effect of KCl and 
MgCl2 on double-stranded and single-stranded DNA, and on the two steps of DNA degradation, 
depurination and subsequent cleavage. We show that KCl and MgCl2 protect both single and 
double-stranded DNA against thermodegradation via the inhibition of depurination. This 
indicates that the protection effect of salt on the thermodegradation of double-stranded DNA is 
not mediated by the stabilization of the double-helix, but by a direct protection of the N-
glycosidic bond at high temperature. Our results also shown that formation of apurinic sites is 
not immediatly followed by DNA cleavage, even at hyperthermophilic temperatures. They 
suggest that the number and nature of heat-induced DNA lesions which have to be repaired might 
be quite different from one hyperthermophile to another, depending of their intracellular salt 
concentration. We plan now to study the effect of compatible solute on DNA stability at high 
temperature in collaboration with our EC partners.


Marguet, E. and Forterre, P. 
 DNA stability at temperatures typical for hyperthermophiles. 
 Nucl. Acid Res. 22, 1681-1686 (1994)
Forterre, P., Bergerat, A. and Lopez-Garcia, P.
 The unique DNA topology and DNA topoisomerases of hyperthermophilic archaea. 
 FEMS Microbiol. Rev. 18, 237-248 (1996) 

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