Identification of the gene encoding archeal‐specific DNA‐binding proteins of the Sac10b family

Sir In the 1980s, several abundant, small and basic DNA-binding proteins were isolated from extreme thermophilic archaea of the genus Sulfolobus (Grote et al., 1986, Biochim Biophys Acta, 873: 405–413). They were grouped into three classes according to their molecular sizes (7, 8 and 10 kDa respectively) (Djick and Reinhardt, 1986, Bacterial Chromatin. Gualerzi and Pon (eds). New York: Springer-Verlag, pp. 185–218). Each of these classes comprises several members that can be discriminated according to their electrophoretic properties: 7a-e, 8a,b and 10a,b. Biochemical and structural characterization of several of these various putative archaeal histone-like proteins, especially those of the 7 kDa and the 10 kDa families (Lurz et al., 1986, EMBO J5: 3715–3721), was first performed in the mid-1980s by Reinhardt and co-workers. Further work concentrated exclusively on two proteins of the 7 kDa group, the protein Sso7d from Sulfolobus solfataricus and Sac7d from Sulfolobus acidocaldarius. Their genes were cloned and sequenced, and their three-dimensional structures were determined by both NMR and X-ray crystallography (Agabeck et al.,1998, Nature Struct Biol5: 579–584; Gao et al., 1998, Nature Struct Biol5: 782–786; Robinson et al., 1998, Nature392: 202–205). These proteins have no sequence similarity with archaeal histones or histone-like proteins discovered in other archaea (Grayling et al., 1994, Syst Appl Microbiol16: 582–590). Both Sac7d and Sso7d contain extensive beta-sheet structures and bind double-stranded DNA with micromolar affinity in a non-co-operative manner (Baumann et al., 1994, Nature Struct Biol1: 808–819; Lundbäck et al., 1998, J Mol Biol276: 775–786). Binding of proteins of this family leads to significant bending and unwinding of DNA and induces negative supercoils, suggesting a role in DNA compaction in Sulfolobus (Lopez-Garcia et al., 1998; Nucleic Acids Res26: 2322–2328). In contrast, the 8 kDa and the 10 kDa proteins have been largely ignored since their discovery, and the genes encoding these proteins are unknown. However, we recently noticed that short internal peptide sequences of 17 and 28 amino acids were reported for the proteins 8b and 10b of S. acidocaldarius (Djick and Reinhardt, 1986, Bacterial Chromatin. Gualerzi and Pon (eds). New York: Springer-Verlag, pp. 185–218). Using these sequences as queries, we searched for the putative genes encoding these two proteins concurrently and found in Sulfolobus shibatae a gene encoding a protein with a region of very high similarity to the peptide isolated from Sac10b. This gene (ssh10b) is predicted to encode a protein of 10 578 Da with a deduced isoelectric point of 11.24. Interestingly, ssh10b is located at the 5′ end of the S. shibatae reverse gyrase topR gene (Jaxel et al., 1996, Nucleic Acids Res24: 4668–4675; Fig. 1). Whereas proteins Sac7d and Sso7d have no homologues in other archaea whose genomes have been completely sequenced, we found an homologue of ssh10b in all archaeal genomes (see alignment in Fig. 2), but not in either bacteria or eukarya. In addition, S. Shibatae was found to have a paralogue of ssh10b that, surprisingly, overlaps the last 245 nucleotides of the topR gene in the reverse orientation (Fig. 1). This gene (ssh10b2) was predicted to encode a protein of 10 258 Da with an isoelectric point of 10.23 (Fig. 2). This protein probably does not correspond to Ssh10a because, according to Djick and Reinhardt (1986), Sac10a and Sac10b are unrelated in terms of amino-acid sequence. A dendrogram of all archaeal protein homologues to Ssh10b shows a clear-cut separation between the protein Ssh10b2 from S. Shibatae and all other archaeal 10b proteins (not shown). . Gene cluster containing the two Sulfolobus shibatae ssh10 genes and the reverse gyrase gene (topR ) (GenBank, X98420). . Alignment of archaeal protein homologues of Sulfolobus acidocaldarius DNA-binding protein Sac10b. Sac10b is the sequenced peptide fragment of Sac10b (Djick and Reinhardt, 1986);ssh10b, Sulfolobus shibatae (GenBank, X98420); ssh10b-2, S. shibatae (GenBank, X98420); afu10b, Archaeoglobus fulgidus (AF1956); afu10b-2, A. fulgidus (AF1067); pho10b, Pyrococcus horikoshi (pHs053); mth10b, Methanobacterium thermoautotrophicum (mt2622599); mja10b, Methanococcus jannasii (MJ0212). Archaeal proteins of the Sac10b family are the first known, small, basic DNA-binding proteins that are both ubiquitous in archae and specific to this domain. This suggests an important physiological role for these proteins. Preliminary biochemical and structural analyses have shown that Sac10b is a dimer in solution and has a greater affinity for DNA than its two counterparts of 7 and 8 kDa (Djick and Reinhardt, 1986, Bacterial Chromatin. Gualerzi and Pon (eds). New York: Springer-Verlag, pp. 185–218). It was shown by electron microscopy that the fixation of Sac10b to DNA is co-operative and that it can envelop two strands of duplex into a helical protein structure in a RecA-like manner (Lurz et al., 1986, EMBO J5: 3715–3721). The protein does not compact DNA, suggesting that it is not involved in packaging. Reinhardt and colleagues proposed instead that Sac10b could be involved in an unknown recombination/repair process in Sulfolobus. The location of the two paralogues of Sac10b on each side of the S. Shibatae reverse gyrase gene is thus intriguing, considering that reverse gyrase might also be involved in DNA recombination and/or repair (Duguet, 1995, Nucleic Acids and Molecular Biology, vol. 9. Eckstein and Lilley (eds). Berlin: Springer, pp. 84–114).