The spindle-shaped virus SSV1 of the hyperthermophilic archaeon Sulfolobus shibatae encodes an integrase (SSV1 Int). Here, the crystal structure of the C-terminal catalytic domain of SSV1 Int is reported. This is the first structural study of an archaeal tyrosine recombinase. Structural comparison shows that the C-terminal domain of SSV1 Int possesses a core fold similar to those of tyrosine recombinases of both bacterial and eukaryal origin, apart from the lack of a conserved helix corresponding to αI of Cre, indicating conservation of these enzymes among all three domains of life. Five of the six catalytic residues cluster around a basic cleft on the surface of the structure and the nucleophile Tyr314 is located on a flexible loop that stretches away from the central cleft, supporting the possibility that SSV1 Int cleaves the target DNA in a trans mode. Biochemical analysis suggests that the N-terminal domain is responsible for the dimerization of SSV1 Int. The C-terminal domain is capable of DNA cleavage and ligation, but at efficiencies significantly lower than those of the full-length protein. In addition, neither the N-terminal domain alone nor the C-terminal domain alone shows a strong sequence preference in DNA binding. Therefore, recognition of the core-type sequence and efficient catalysis by SSV1 Int presumably requires covalent linkage and interdomain communication between the two domains.

Archaeal chromatin proteins Cren7 and Sul7d from Sulfolobus are DNA benders. To better understand their architectural roles in chromosomal DNA organization, we analyzed DNA compaction by Cren7 and Sis7d, a Sul7d family member, from Sulfolobus islandicus at the single-molecule (SM) level by total single-molecule internal reflection fluorescence microscopy (SM-TIRFM) and atomic force microscopy (AFM). We show that both Cren7 and Sis7d were able to compact singly tethered λ DNA into a highly condensed structure in a three-step process and that Cren7 was over an order of magnitude more efficient than Sis7d in DNA compaction. The two proteins were similar in DNA bending kinetics but different in DNA condensation patterns. At saturating concentrations, Sis7d formed randomly distributed clusters whereas Cren7 generated a single and highly condensed core on plasmid DNA. This observation is consistent with the greater ability of Cren7 than of Sis7d to bridge DNA. Our results offer significant insights into the mechanism and kinetics of chromosomal DNA organization in Crenarchaea. IMPORTANCE A long-standing question is how chromosomal DNA is packaged in Crenarchaeota, a major group of archaea, which synthesize large amounts of unique small DNA-binding proteins but in general contain no archaeal histones. In the present work, we tested our hypothesis that the two well-studied crenarchaeal chromatin proteins Cren7 and Sul7d compact DNA by both DNA bending and bridging. We show that the two proteins are capable of compacting DNA, albeit with different efficiencies and in different manners, at the single molecule level. We demonstrate for the first time that the two proteins, which have long been regarded as DNA binders and benders, are able to mediate DNA bridging, and this previously unknown property of the proteins allows DNA to be packaged into highly condensed structures. Therefore, our results provide significant insights into the mechanism and kinetics of chromosomal DNA organization in Crenarchaeota.

SSV-type integrases, encoded by fuselloviruses which infect the hyperthermophilic archaea of the Sulfolobales, are archaeal members of the tyrosine recombinase family. These integrases catalyze viral integration into and excision from a specific site on the host genome. In the present study, we have established an in vitro integration/excision assay for SSV2 integrase (Int SSV2 ). Int SSV2 alone was able to catalyze both integration and excision reactions in vitro. A 27-bp specific DNA sequence is minimally required for the activity of the enzyme, and its flanking sequences influence the efficiency of integration by the enzyme in a sequence-nonspecific manner. The enzyme forms a tetramer through interactions in the N-terminal part (residues 1 to 80), interacts nonspecifically with DNA and performs chemical catalysis in the C-terminal part (residues 165 to 328), and appears to recognize and bind the specific site of recombination in the middle portion (residues 81 to 164). It is worth noting that an N-terminally truncated mutant of Int SSV2 (residues 81 to 328), which corresponded to the putative product of the 3=-end sequence of the Int SSV2 gene of the integrated SSV2 genome, was unable to form tetramers but possessed all the catalytic properties of full-length Int SSV2 except for the slightly reduced recombination activity. Our results suggest that, unlike integrase, SSV-type integrases alone are capable of catalyzing viral DNA recombination with the host genome in a simple and reversible fashion. IMPORTANCE Archaea are host to a variety of viruses. A number of archaeal viruses are able to integrate their genome into the host genome.Many known archaeal viral integrases belong to a unique type, or the SSV type, of tyrosine recombinases. SSV-type integrases catalyze viral integration into and excision from a specific site on the host genome. However, the molecular details of the recombination process have yet to be fully understood because of the lack of an established in vitro recombination assay system. Here we report an in vitro assay for integration and excision by SSV2 integrase, a member of the SSV-type integrases. We show that SSV2 integrase alone is able to catalyze both integration and excision and reveal how different parts of the target DNA and the enzyme serve their roles in these processes. Therefore, our results provide mechanistic insights into a simple recombination process catalyzed by an archaeal integrase. T yrosine recombinases (i.e., members of the integrase family), named after the nucleophile tyrosine residue in the enzymes that forms a transient 3=-phosphotyrosine covalent linkage to the DNA substrate in the reaction intermediate, are widespread in all three domains of life (1, 2). They catalyze recombination reactions in many important biological processes in both prokaryotes and eukaryotes, such as integration and excision of a phage genome into and out of the host genome ( Int and HP1 Int), maintenance of plasmid copy number (Flp), resolution of replicon dimers into monomers (C...

The accuracy of DNA synthesis by DNA polymerase B1 from the hyperthermophilic archaeon Sulfolobus solfataricus (Sso pol B1) at near the physiological temperature was investigated using M13-based mutational assays. Sso pol B1 showed replication fidelity similar to or higher than most viral, bacterial, and eukaryotic replicases. The fidelity of the enzyme was about three times as high at 70 degrees C as at 55 degrees C. Approximately two-thirds of the errors made by the enzyme were single-base substitutions, of which 58% were C --> T transition. Frameshift mutations, mostly resulting from single-base deletions, accounted for 19% of the total errors. An exonuclease-deficient mutant of Sso pol B1 was three times as mutagenic as the wild-type enzyme, suggesting that the intrinsic proofreading function contributed only modestly to the fidelity of the enzyme. Kinetic assays showed that the frequencies of all possible misincorporations by an exonuclease-deficient triple-point mutant of Sso pol B1 ranged from 5.4 x 10(-5) to 4.6 x 10(-4). The high fidelity of this enzyme in DNA synthesis was based primarily on K (m) difference rather than V (max) difference. These properties of Sso pol B1 are consistent with the proposed role of the enzyme as a replicase in S. solfataricus.

Much of our knowledge in conventional biochemistry has derived from bulk assays. However, many stochastic processes and transient intermediates are hidden when averaged over the ensemble. The powerful technique of single-molecule fluorescence microscopy has made great contributions to the understanding of life processes that are inaccessible when using traditional approaches. In single-molecule studies, quantum dots (Qdots) have several unique advantages over other fluorescent probes, such as high brightness, extremely high photostability, and large Stokes shift, thus allowing long-time observation and improved signal-to-noise ratios. So far, however, there is no convenient way to label proteins purified from budding yeast with Qdots. Based on BirA–Avi and biotin–streptavidin systems, we have established a simple method to acquire a Qdot-labeled protein and visualize its interaction with DNA using total internal reflection fluorescence microscopy. For proof-of-concept, we chose replication protein A (RPA) and origin recognition complex (ORC) as the proteins of interest. Proteins were purified from budding yeast with high biotinylation efficiency and rapidly labeled with streptavidin-coated Qdots. Interactions between proteins and DNA were observed successfully at the single-molecule level.

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The fluorescence microscopy has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. In single molecule assays for studying DNA-protein interactions, there are two important factors for consideration: the DNA substrate with enough length for easy observation and labeling a protein with a suitable fluorescent probe. 48.5 kb λ DNA is a good candidate for the DNA substrate. Quantum dots (Qdots), as a class of fluorescent probes, allow long-time observation (minutes to hours) and high-quality image acquisition. In this paper, we present a protocol to study DNA-protein interactions at the single-molecule level, which includes preparing a site-specifically modified λ DNA and labeling a target protein with streptavidin-coated Qdots. For a proof of concept, we choose ORC (origin recognition complex) in budding yeast as a protein of interest and visualize its interaction with an ARS (autonomously replicating sequence) using TIRFM. Compared with other fluorescent probes, Qdots have obvious advantages in single molecule studies due to its high stability against photobleaching, but it should be noted that this property limits its application in quantitative assays.

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