We investigated the capacity of the hyperthermophile Pyrococcus furiosus for DNA repair by measuring survival at high levels of 60 Co ␥-irradiation. The P. furiosus 2-Mb chromosome was fragmented into pieces ranging from 500 kb to shorter than 30 kb at a dose of 2,500 Gy and was fully restored upon incubation at 95°C. We suggest that recombination repair could be an extremely active repair mechanism in P. furiosus and that it might be an important determinant of survival of hyperthermophiles at high temperatures.Pyrococcus furiosus is a hyperthermophile growing optimally at 100°C. It is a member of the Archaea, a group of prokaryotes which have many molecular features in common with modern eukaryotes, and which are thought by many to be ancestral life forms (25). The exceptional degree of tolerance and resistance of hyperthermophiles to high temperatures must include adaptations that affect all levels of the cellular machinery, including the enzymes that are involved in maintaining the integrity and stability of genomic DNA. Kopylov et al. (14) reported that a closely related member of the Archaea, Thermococcus stetteri, is 12 times more resistant to ␥-irradiation than Escherichia coli but 2 times more sensitive than the bacterium Deinococcus radiodurans. In addition, Peak et al. (20) have shown that at 100°C, the DNA of P. furiosus is 20 times more resistant to thermal breakage in vivo than the DNA from the mesophile E. coli. Thermophilic members of the Archaea have histone proteins (24) known to give partial protection of plasmid DNA to fast neutrons and ␥-photons (12) and to prevent thermal denaturation of DNA (5). However, the protective effect of DNA-binding proteins cannot account for the extreme resistance of these microorganisms to heat and ␥-irradiation (12,14,20), suggesting the presence of very active mechanisms for DNA repair.Among the lesions induced by ionizing radiation in cellular DNA, double-strand breaks (DSBs) are the least efficiently repaired, and their frequency is correlated with cell death (9). Indeed, E. coli and most other organisms cannot survive if more than two or three DSBs are introduced per chromosome, independently of their physiological state (15,21). DSBs are noninformative lesions that affect the DNA double helix at the same site, eliminating intact template for repair and precluding any excision repair processes. However, D. radiodurans has been found to be extremely resistant to ionizing radiation (19). This organism can repair more than 100 DSBs per chromosome, induced by ionizing radiation, without loss of viability (19). Its extreme resistance to ␥-irradiation is attributed to possible adaptation to desiccation (16). RecA-dependent recombinational repair and single-strand annealing are the two mechanisms proposed to account for repair of ionizing-radiation damage in D. radiodurans (16,18).In contrast, there is currently very little information about DNA repair systems in hyperthermophiles, and what information there is consists mainly of the characterization of DNA rep...