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Characterization of SsoSSB, Sso1450, Sso2001 Proteins and Analysis of CRISPR and cas Genes from Sulfolobus solfataricus P2
(2008)
- Following the complete sequencing of the genome of Sulfolobus solfataricus (Sso) P2, this organism has been widely used as a model strain for crenarchaea. The present work concentrated on the characterization of a newly discovered single-stranded binding (SSB) protein from Sso P2 and the computational and experimental analyses of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (cas) genes, respectively. The DNA-binding properties of SsoSSB, the organization of the CRISPR loci and the biochemical properties of some of the cas gene products of Sso P2 were studied. Size-exclusion chromatography indicated that SsoSSB exists as a monomer in solution. Using fluorescence anisotropy as a method to study the interaction of SsoSSB to DNA, it can be shown that SsoSSB binds as a monomer to small oligonucleotides. The approximate binding site size is 4- 6 nt per protein molecule as determined by native electrophoresis. SsoSSB shows a more than 10 fold higher binding affinity to single-stranded (ss) DNA as compared to double-stranded (ds) DNA, which is consistent with former reports. The dissociation constant could be determined to be in a low nanomolar range. Furthermore, SsoSSB preferentially binds to pyrimidine-rich ssDNA as compared to purine-rich DNA. This property is similar to that observed for human replication protein A (RPA). The clustering of repeat sequences in CRISPR loci and the associated cas genes have emerged recently as a new genomic feature of Archaea and of some Bacteria. Formerly, the cas genes have been predicted to encode repair proteins. In the present work, five CRISPR loci and their cas genes in Sso P2 were analyzed with respect to their genomic organization. The repeats of the CRISPR loci show highly conserved sequences at regular intervals, separated by spacer sequences of similar size. Most of the cas gene groups flanking a CRISPR locus contain homologous genes that were also found at other CRISPR loci. The cas genes could be grouped by gene location and gene order. Mostly, in each group they were head-to-tail arranged implying a functional relation. The operons of the cas genes sso1996-2002, sso1438-1443 and sso1398-1403 could be shown to contain Transcription Factor B recognition element (BRE), TATA-box, Shine-Dalgarno and terminator sequences specific for Sulfolobus. Most cas genes could not be expressed in a soluble form in E.coli, even when the expression conditions were widely varied. Refolding of the insoluble proteins was then undertaken. sso1442, sso1996 and sso1997 could be expressed in partially soluble form in E.coli, however catalytical activities could not be identified for these proteins. Refolding of Sso1999, a putative helicase, yielded a soluble protein. However no helicase and ATPase activity could be detected in the renatured Sso1999. Defined biochemical activities could be only assigned to the proteins Sso1450 and Sso2001. In the latter case, the sso2001 gene was fused with an esterase gene from Alicyclobacillus acidocaldarius, and was co-expressed in a soluble form. The enzymatic screening indicated that Sso2001 harbored a nuclease activity. Further experiments showed that Sso2001 was an endonuclease with specificity for cleavage near G residues. The nuclease activity was optimal at the neutral pH range with another activity peak at pH 3. Specific point mutations introduced in Sso2001 indicate that this protein was not a HD-family nuclease as previously predicted. The protein Sso1450 (COG1518), which is considered to be a marker protein of the CRISPR and Cas system, bound nucleic acids, including ssDNA, dsDNA and RNA, with high affinity. The dissociation constant of binding to DNA oligonucleotides was in the nanomolar range. EMSA experiments indicated an aggregation of Sso1450 on the DNA substrates. Interestingly, Sso1450 promoted the annealing of complementary ssDNAs. This finding supported a role of Sso1450 in the recombination of repeat sequences of the CRISPR system as suggested by Koonin’s group (Makarova et al., 2006). The CRISPRs were thought to play a major role in a newly discovered genome immune system in prokaryotes.
