Abstract
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.
Highlights
Archaeal chromatin proteins Cren7 and Sul7d from Sulfolobus are DNA benders
We investigated the architectural roles of Cren7 and Sis7d, a Sul7d family protein from S. islandicus, in chromosomal organization using singlemolecule total internal reflection fluorescence microscopy (SM-TIRFM) and atomic force microscopy (AFM)
We found that DNA was well extended at a flow rate of 100 l/min (Fig. 1, top panels)
Summary
Archaeal chromatin proteins Cren and Sul7d from Sulfolobus are DNA benders. To better understand their architectural roles in chromosomal DNA organization, we analyzed DNA compaction by Cren 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). Present in a number of archaeal phyla These proteins compact DNA in a manner similar to that seen with eukaryotic histones, wrapping DNA around the core tetramer/ hexamer to form nucleosome-like or filamentous structures [2, 3]. Proteins of the Sac10b family, known as Alba, are highly conserved in Archaea Members of this protein family from Sulfolobus appear to be able to bind RNA in vivo [10, 11], they were shown previously to compact DNA in vitro [12]. A single-molecule (SM) analysis performed using magnetic tweezers revealed that Cren and Sso7d compact DNA to similar extents by introducing rigid bends [20] These results are consistent with the notion that the two proteins are DNA benders. It is of interest to determine if and how Cren and Sul7d serve distinct roles in chromosomal organization
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