An Abundant DNA Binding Protein from the Hyperthermophilic Archaeon Sulfolobus shibatae Affects DNA Supercoiling in a Temperature-Dependent Fashion

Abstract

The DNA binding protein Ssh10b, a member of the Sac10b family, has been purified from the hyperthermophilic archaeon Sulfolobus shibatae. Ssh10b constitutes about 4% of the cellular protein. Electrophoretic mobility shift assays showed that Ssh10b first bound a double-stranded DNA fragment with an estimated binding size of ϳ ϳ12 bp, forming distinct shifts, until the DNA was coated with the protein. Binding of more Ssh10b resulted in the formation of smears of lower mobilities. The migration pattern of the smearing Ssh10b-DNA complexes was affected by temperature, whereas that of complexes associated with the distinct shifts was not. Interestingly, Ssh10b was capable of constraining negative DNA supercoils in a temperaturedependent fashion. While the ability of the protein to constrain supercoils was weak at 25°C, it was enhanced substantially at 45°C or higher temperatures (up to 80°C). Taken together, our data suggest that archaeal proteins of the Sac10b family may affect the topology of chromosomal DNA in thermophilic archaea at their growth temperatures.

The neomuran origin of archaebacteria, the negibacterial root of the universal tree and bacterial megaclassification.

“…Rather than being a novel archaebacterial enzyme, a pre-existing one simply switched its specificity ; I suggest that the switch was from queuine insertion to archaeosine insertion. A more convincingly unique archaebacterial protein is the small, 10 kDa DNA-binding protein, 10b ; in Sulfolobus, its binding produces passive negative supercoils at very high temperatures, but not at mesic ones (Xue et al, 2000). I suggest that it evolved as a secondary adaptation to hyperthermophily in the ancestral archaebacterium, as a substitute for active negative supercoiling by DNA gyrase, which was lost in the neomuran cenancestor (i.e.…”

“…I suggest that, as one thermophilic stem neomuran lineage adapted to hyperthermophily, thereby becoming the archaebacterial cenancestor, it lost H1 and evolved DNA-binding protein 10b, which makes negative supercoils in Sulfolobus only at very high temperatures (Xue et al, 2000), when adapting to hyperthermophily. At the same time, reverse gyrase, found in hyperthermophiles, evolved to reduce the risk of denaturation of DNA at high temperature by supercoiling it positively.…”

The Archaeal Sac10b Protein Family: Conserved Proteins with Divergent Functions

“…Sac10b itself was the first DNA-binding protein of the hyperthermophile Sulfolobus acidocaldaricus studied in detail, therefore its name was used as the family name [10]. Sac10b homologues have 87 to 102 amino acid residues and share ~35 to 66 % identity and 55 to 82 % similarity at the amino acid sequence level [22]. Multiple alignment of the Sac10b proteins shows that they possess a positive charge density of ~15 to16 % and share several fairly conserved charged positions [43].…”

“…In 1999, Lou et al isolated Ssh12, an abundant DNA-binding protein of the archaeon Sulfolobus shibatae [21]. Later, Ssh12 was proved to be Ssh10b, a member of the Sac10b protein family [22,23]. Identification of the gene encoding Ssh10b and finding homologues of Ssh10b occurred in all archaeal genomes sequenced at that time but not in bacteria and eukaryota [24], stimulated a large number of biophysical, biochemical, structural and physiological studies of these proteins.…”

Crystal Structure and Functional Analysis of an Archaeal Chromatin Protein Alba from the Hyperthermophilic ArchaeonPyrococcus horikoshiiOT3

“…They are small, basic proteins, and they occur as a dimeric form in solution. 4) Since Alba binds nonspecifically to doublestranded DNA and is predominantly present in chromatin, 4,5) it is thought to play a major role in the architecture of the archaeal chromosome. It was further found that the Alba protein (SsoAlba) from Sulfolobus solfataricus forms a stable complex with an archaeal Sir2 homolog from S. solfataricus, which has histone deacetylase activity, and that acetylation of SsoAlba at Lys16 strongly reduces the affinity of the protein for DNA.…”