The following text describes a protein:
Function:
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. 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. 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. Has also been shown to have no detectable guide sequence-independent dsDNase activity. 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); Involved in defense against invading mobile genetic elements. 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. 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. Positions 4 to 16 of the tDNA need to be base paired to the gDNA for efficient tDNA cleavage. 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; 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.
Domain:
Has 4 domains (N-terminal, PAZ, Mid and PIWI). The N-terminal and PAZ domains are joined by linker L1, the PAZ and Mid domains are joined by linker L2. The domains assemble in 2 lobes; the PAZ lobe consists of the N-terminal, L1, PAZ and L2 domains, while the PIWI lobe has the Mid and PIWI domains. The PIWI domain assumes an RNase H fold and has the catalytic residues. gDNA lies between the 2 lobes, with its unpaired 5'-end anchored in the Mid lobe while the 3'-end anchors in the PAZ domain in the absence of target or if target nucleic acid has not fully base-paired with the gDNA. The N-terminal, Mid and PAZ domains alter position upon binding different length gDNA, forming and closing DNA-binding channels. The 2 lobes and gDNA move considerably upon formation of the ternary gDNA:target RNA:TtAgo complex to accomodate and base pair the target RNA. Deletions of the N-terminus (residues 1-106 or 1-177) have lost catalytic activity. Upon release of the 3'-end of the gDNA from the PAZ domain during nucleic acid duplex formation, Glu-512 moves about 13 Angstroms to complete the active site.
Sequence:
MNHLGKTEVFLNRFALRPLNPEELRPWRLEVVLDPPPGREEVYPLLAQVARRAGGVTVRMGDGLASWSPPEVLVLEGTLARMGQTYAYRLYPKGRRPLDPKDPGERSVLSALARRLLQERLRRLEGVWVEGLAVYRREHARGPGWRVLGGAVLDLWVSDSGAFLLEVDPAYRILCEMSLEAWLAQGHPLPKRVRNAYDRRTWELLRLGEEDPKELPLPGGLSLLDYHASKGRLQGREGGRVAWVADPKDPRKPIPHLTGLLVPVLTLEDLHEEEGSLALSLPWEERRRRTREIASWIGRRLGLGTPEAVRAQAYRLSIPKLMGRRAVSKPADALRVGFYRAQETALALLRLDGAQGWPEFLRRALLRAFGASGASLRLHTLHAHPSQGLAFREALRKAKEEGVQAVLVLTPPMAWEDRNRLKALLLREGLPSQILNVPLREEERHRWENALLGLLAKAGLQVVALSGAYPAELAVGFDAGGRESFRFGGAACAVGGDGGHLLWTLPEAQAGERIPQEVVWDLLEETLWAFRRKAGRLPSRVLLLRDGRVPQDEFALALEALAREGIAYDLVSVRKSGGGRVYPVQGRLADGLYVPLEDKTFLLLTVHRDFRGTPRPLKLVHEAGDTPLEALAHQIFHLTRLYPASGFAFPRLPAPLHLADRLVKEVGRLGIRHLKEVDREKLFFV