“…The molecule carries a biotin label at the 5′ end of one strand and a DIG label at the 5′ end of the complementary strand (Figure 1A). For control experiments, equivalent PCR products were generated from pGB1/S1 and pSB1 (34), which have, respectively, one or no recognition site for SfiI, to give 1- or 0-site constructs (Figure 1A). Figure 1.Schematics TPM experiments.…”
Many proteins that interact with DNA perform or enhance their specific functions by binding simultaneously to multiple target sites, thereby inducing a loop in the DNA. The dynamics and energies involved in this loop formation influence the reaction mechanism. Tethered particle motion has proven a powerful technique to study in real time protein-induced DNA looping dynamics while minimally perturbing the DNA–protein interactions. In addition, it permits many single-molecule experiments to be performed in parallel. Using as a model system the tetrameric Type II restriction enzyme SfiI, that binds two copies of its recognition site, we show here that we can determine the DNA–protein association and dissociation steps as well as the actual process of protein-induced loop capture and release on a single DNA molecule. The result of these experiments is a quantitative reaction scheme for DNA looping by SfiI that is rigorously compared to detailed biochemical studies of SfiI looping dynamics. We also present novel methods for data analysis and compare and discuss these with existing methods. The general applicability of the introduced techniques will further enhance tethered particle motion as a tool to follow DNA–protein dynamics in real time.
“…The molecule carries a biotin label at the 5′ end of one strand and a DIG label at the 5′ end of the complementary strand (Figure 1A). For control experiments, equivalent PCR products were generated from pGB1/S1 and pSB1 (34), which have, respectively, one or no recognition site for SfiI, to give 1- or 0-site constructs (Figure 1A). Figure 1.Schematics TPM experiments.…”
Many proteins that interact with DNA perform or enhance their specific functions by binding simultaneously to multiple target sites, thereby inducing a loop in the DNA. The dynamics and energies involved in this loop formation influence the reaction mechanism. Tethered particle motion has proven a powerful technique to study in real time protein-induced DNA looping dynamics while minimally perturbing the DNA–protein interactions. In addition, it permits many single-molecule experiments to be performed in parallel. Using as a model system the tetrameric Type II restriction enzyme SfiI, that binds two copies of its recognition site, we show here that we can determine the DNA–protein association and dissociation steps as well as the actual process of protein-induced loop capture and release on a single DNA molecule. The result of these experiments is a quantitative reaction scheme for DNA looping by SfiI that is rigorously compared to detailed biochemical studies of SfiI looping dynamics. We also present novel methods for data analysis and compare and discuss these with existing methods. The general applicability of the introduced techniques will further enhance tethered particle motion as a tool to follow DNA–protein dynamics in real time.
“…Such tetrameric REases bind two target DNA copies and generate double-strand breaks at both sites cutting four phosphodiester bonds simultaneously (8–11). Tetramerization provides stabilization of the functional REase dimer (12,13) and increase the specificity of target site recognition (14). A few REases assemble into transient oligomeric intermediates to achieve DNA double-strand breaks.…”
To cut DNA at their target sites, restriction enzymes assemble into different oligomeric structures. The Ecl18kI endonuclease in the crystal is arranged as a tetramer made of two dimers each bound to a DNA copy. However, free in solution Ecl18kI is a dimer. To find out whether the Ecl18kI dimer or tetramer represents the functionally important assembly, we generated mutants aimed at disrupting the putative dimer–dimer interface and analysed the functional properties of Ecl18kI and mutant variants. We show by atomic force microscopy that on two-site DNA, Ecl18kI loops out an intervening DNA fragment and forms a tetramer. Using the tethered particle motion technique, we demonstrate that in solution DNA looping is highly dynamic and involves a transient interaction between the two DNA-bound dimers. Furthermore, we show that Ecl18kI cleaves DNA in the synaptic complex much faster than when acting on a single recognition site. Contrary to Ecl18kI, the tetramerization interface mutant R174A binds DNA as a dimer, shows no DNA looping and is virtually inactive. We conclude that Ecl18kI follows the association model for the synaptic complex assembly in which it binds to the target site as a dimer and then associates into a transient tetrameric form to accomplish the cleavage reaction.
“…The ability of WT AfAgo to form homodimers and bring two nucleic acid fragments into close proximity, to the best of our knowledge, is unique among Argonaute proteins, and raises additional questions regarding the currently unknown AfAgo function. Simultaneous interaction with two target sites in the case of restriction endonucleases is believed to increase specificity by preventing inadvertent cleavage of lone unmodified target sites [23, 29]. However, since AfAgo has no intrinsic nuclease activity, it cannot be directly involved in host defense against invading nucleic acids, as recently proposed for the catalytically active full-length pAgos [30, 31].…”
14Background: Argonaute (Ago) proteins are found in all three domains of life. The best characterized 15 group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood 16 prokaryotic Ago (pAgo) proteins are full-length pAgos. They are monomeric proteins, all composed of 17 four major structural/functional domains (N, PAZ, MID and PIWI) and thereby closely resemble eAgos.
18It is believed that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain 19 performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter 20 and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism 21 and function remain unknown.
22Results: In this work we focus on AfAgo, a short pAgo protein encoded by an archaeon 23 Archaeoglobus fulgidus. We find that in all previously solved AfAgo structures, its two monomers form 24 substantial dimerization interfaces involving the C-terminal β-sheets. Led by this finding, we have 25 employed various biochemical and biophysical assays, including single-molecule FRET, SAXS and 26 AFM, to test the possible dimerization of AfAgo. SAXS results confirm that WT AfAgo, but not the 27 dimerization surface mutant AfAgoΔ, forms a homodimer both in the apo-form and when bound to a 28 nucleic acid. Single molecule FRET and AFM studies demonstrate that the dimeric WT AfAgo binds 29 two ends of a linear DNA fragment, forming a relatively stable DNA loop.
30Conclusion: Our results show that contrary to other characterized Ago proteins, AfAgo is a stable 31 homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This 32 finding broadens the range of currently known Argonaute-nucleic acid interaction mechanisms. Argonaute (Ago) proteins are found in all three domains of life (bacteria, archaea, and eukaryotes).
37The best characterized group is eukaryotic Ago (eAgo) proteins. Being the functional core of RNA 38 interference machinery, eAgos are involved in regulation of gene expression, silencing of mobile 39 genome elements, and defense against viruses. From the structural and mechanistic point of view, all 40 eAgos are very similar, as they all use small RNA molecules as guides for sequence-specific 41 recognition of RNA targets, and are monomeric proteins sharing four conserved functional domains, 42 which are organized in a bilobed structure [1]. The N-terminal lobe consists of the N-domain that 43 separates guide and target strands [2], and the PAZ domain responsible for binding the 3′-terminus of 44 the guide RNA; the C-terminal lobe consists of the MID domain, which binds the 5′-terminus of the 45 guide RNA, and the PIWI domain, an RNase. Upon recognition of the RNA target, eAgos may either 46 cleave it employing the catalytic activity of the PIWI domain, or, especially eAgo proteins that encode 47 catalytically inactive PIWI domains, recruit partner proteins leading to degradation of the target RNA 48 or repression of its translation ...
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