View source for 6wdz


==Porcine circovirus 2 Rep domain complexed with a single-stranded DNA 10-mer comprising the cleavage site==
<StructureSection load='6wdz' size='340' side='right'caption='[[6wdz]], [[Resolution|resolution]] 2.03&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[6wdz]] is a 7 chain structure with sequence from [https://en.wikipedia.org/wiki/Porcine_circovirus_2 Porcine circovirus 2]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6WDZ OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=6WDZ FirstGlance]. <br>
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.03&#8491;</td></tr>
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=EDO:1,2-ETHANEDIOL'>EDO</scene>, <scene name='pdbligand=MN:MANGANESE+(II)+ION'>MN</scene></td></tr>
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=6wdz FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6wdz OCA], [https://pdbe.org/6wdz PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=6wdz RCSB], [https://www.ebi.ac.uk/pdbsum/6wdz PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=6wdz ProSAT]</span></td></tr>
</table>
== Function ==
[https://www.uniprot.org/uniprot/REP_PCV2 REP_PCV2] Essential for the replication of viral ssDNA. The closed circular ssDNA genome is first converted to a superhelical dsDNA. Rep and/or Rep' binds a specific hairpin at the genome origin of replication. Introduces an endonucleolytic nick within the conserved sequence 5'-AGTATTAC-3' in the intergenic region of the genome, thereby initiating the rolling circle replication (RCR). Following cleavage, binds covalently to the 5'-phosphate of DNA as a tyrosyl ester. The cleavage gives rise to a free 3'-OH that serves as a primer for the cellular DNA polymerase. The polymerase synthesizes the (+) strand DNA by rolling circle mechanism. After one round of replication, a Rep-catalyzed nucleotidyl transfer reaction releases a circular single-stranded virus genome, thereby terminating the replication. Displays origin-specific DNA cleavage, and nucleotidyl transferase.<ref>PMID:12954212</ref> <ref>PMID:25768890</ref> 
<div style="background-color:#fffaf0;">
== Publication Abstract from PubMed ==
Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.

Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering.,Tompkins KJ, Houtti M, Litzau LA, Aird EJ, Everett BA, Nelson AT, Pornschloegl L, Limon-Swanson LK, Evans RL, Evans K, Shi K, Aihara H, Gordon WR Nucleic Acids Res. 2021 Jan 25;49(2):1046-1064. doi: 10.1093/nar/gkaa1248. PMID:33410911<ref>PMID:33410911</ref>

From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
</div>
<div class="pdbe-citations 6wdz" style="background-color:#fffaf0;"></div>
== References ==
<references/>
__TOC__
</StructureSection>
[[Category: Large Structures]]
[[Category: Porcine circovirus 2]]
[[Category: Evans III RL]]
[[Category: Gordon WR]]
[[Category: Litzau LA]]
[[Category: Nelson A]]
[[Category: Shi K]]
[[Category: Tompkins K]]