4ncb
Structure of Thermus thermophilus Argonaute bound to guide DNA and 19-mer target DNA with Mg2+
Structural highlights
FunctionAGO_THET2 A DNA-guided ssDNA endonuclease. Uses short ssDNA sequences as guides (gDNA, also called small interfering DNA, siDNA) to bind complementary DNA target strands, resulting in cleavage of the target DNA (tDNA). The cleavage site is 10 nucleotides (nt) downstream of the target residue base-paired with the 5'-end of the gDNA (PubMed:24531762, PubMed:28911094). Plays a role in completion of DNA replication, participates in decatenating replicated DNA and plasmid. In situ purifies with 5'-phosphorylated long DNA (about 1160 nt, maps to the whole chromosome and plasmid), 25-35 nt RNAs that map to the whole chromosome and 15-18 nt DNA that maps to the replication terminus region (ter) on the chromosome and plasmid. Most short DNA starts with dC (PubMed:32846159). Has been shown to have guide sequence-independent dsDNase activity called 'chopping', which requires unstable DNA (high AT-content, multiple mismatches or low salt conditions), and could be used to generate gDNA. Preferentially binds tDNA with dC at its 3'-terminus (PubMed:28262506). Has also been shown to have no detectable guide sequence-independent dsDNase activity (PubMed:32846159). The latter study proposes TtAgo may acquire gDNA from nicked dsDNA, by binding to 5'-phosphorylated-dC nicks, then cleaving 10 nt away on the opposite strand; subsequently an exonuclease (maybe AddA-AddB helicase/nuclease) trims the ends to generate the gDNA (Probable) (PubMed:32846159).[1] [2] [3] [4] [5] Involved in defense against invading mobile genetic elements (PubMed:24531762, PubMed:25331432, PubMed:25902012). TtAgo interferes with plasmid DNA, stimulates expression of specific endogenous genes, including various CRISPR loci and at least part of the CRISPR adaptation machinery, but only when exogenous plasmid DNA is present (PubMed:25902012). Upon purification from E.coli associates with gDNA 13-25 nt long with 5'-phosphorylated ends and with 10-150 nt RNA with 5'-OH. DNA corresponds to the expression plasmid rather than chromosomal DNA; 89% of gDNA starts with dC and 72% has dA in the second position. Endonucleolytically cleaves tDNA with 5'-phosphorylated gDNA but not 5'-phosphorylated gRNA; the active site is involved in processing or binding of ssDNA. Nicks or linearizes supercoiled plasmid target when it has the appropriate gDNA sequences, does not cleave linear tDNA (PubMed:24531762). Positions 4 to 16 of the tDNA need to be base paired to the gDNA for efficient tDNA cleavage (PubMed:19812667). Although the system can support single nucleotide insertions in either the gDNA or tDNA, in all cases cleavage activity is reduced, with a wide range of sequence- and position-specific effects (PubMed:28911094).[6] [7] [8] [9] [10] First characterized as a DNA-guided RNA endonuclease. Uses gDNA to bind complementary RNA target strands, resulting in cleavage of the target RNA. The cleavage site is 10 nucleotides (nt) downstream of the target residue base-paired with the 5'-end of the guide DNA.[11] [12] [13] [14] Publication Abstract from PubMedWe report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5'-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 A have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg2+ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand. Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage.,Sheng G, Zhao H, Wang J, Rao Y, Tian W, Swarts DC, van der Oost J, Patel DJ, Wang Y Proc Natl Acad Sci U S A. 2013 Dec 27. PMID:24374628[15] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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