Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering
Abstract 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...
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| Veröffentlicht in: | Nucleic acids research 2021-01, Vol.49 (2), p.1046-1064 |
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| creator | Tompkins, Kassidy J Houtti, Mo Litzau, Lauren A Aird, Eric J Everett, Blake A Nelson, Andrew T Pornschloegl, Leland Limón-Swanson, Lidia K Evans, Robert L Evans, Karen Shi, Ke Aihara, Hideki Gordon, Wendy R |
| description | Abstract
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. |
| doi_str_mv | 10.1093/nar/gkaa1248 |
| format | Article |
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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.</description><identifier>ISSN: 0305-1048</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/gkaa1248</identifier><identifier>PMID: 33410911</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Amino Acid Motifs ; Amino Acid Sequence ; BASIC BIOLOGICAL SCIENCES ; Circoviridae - enzymology ; Conserved Sequence ; Crystallography, X-Ray ; Deoxyribonuclease I - chemistry ; Deoxyribonuclease I - metabolism ; DNA Helicases - chemistry ; DNA Helicases - metabolism ; DNA, Single-Stranded - chemistry ; DNA, Single-Stranded - metabolism ; Gene Library ; Massively Parallel (Deep) Sequencing ; Models, Molecular ; Molecular Docking Simulation ; Molecular Sequence Data ; Nucleic Acid Conformation ; Nucleic Acid Enzymes ; Nucleic Acid Enzymology ; Plant Viruses - enzymology ; Protein Binding ; Protein Conformation ; Protein Engineering - methods ; Recombinant Fusion Proteins - chemistry ; Recombinant Fusion Proteins - metabolism ; Replication Origin ; Sequence Alignment ; Sequence Homology, Amino Acid ; Single-Strand Specific DNA and RNA Endonucleases - chemistry ; Single-Strand Specific DNA and RNA Endonucleases - metabolism ; Substrate Specificity ; Trans-Activators - chemistry ; Trans-Activators - metabolism ; Viral Proteins - chemistry ; Viral Proteins - metabolism</subject><ispartof>Nucleic acids research, 2021-01, Vol.49 (2), p.1046-1064</ispartof><rights>The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. 2021</rights><rights>The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c509t-e0428fcb1513d8fa603b94b1680879a1708555b9262ad1cac82262c6291e30423</citedby><cites>FETCH-LOGICAL-c509t-e0428fcb1513d8fa603b94b1680879a1708555b9262ad1cac82262c6291e30423</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826260/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826260/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,1598,27903,27904,53769,53771</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/33410911$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://www.osti.gov/biblio/1759119$$D View this record in Osti.gov$$Hfree_for_read</backlink></links><search><creatorcontrib>Tompkins, Kassidy J</creatorcontrib><creatorcontrib>Houtti, Mo</creatorcontrib><creatorcontrib>Litzau, Lauren A</creatorcontrib><creatorcontrib>Aird, Eric J</creatorcontrib><creatorcontrib>Everett, Blake A</creatorcontrib><creatorcontrib>Nelson, Andrew T</creatorcontrib><creatorcontrib>Pornschloegl, Leland</creatorcontrib><creatorcontrib>Limón-Swanson, Lidia K</creatorcontrib><creatorcontrib>Evans, Robert L</creatorcontrib><creatorcontrib>Evans, Karen</creatorcontrib><creatorcontrib>Shi, Ke</creatorcontrib><creatorcontrib>Aihara, Hideki</creatorcontrib><creatorcontrib>Gordon, Wendy R</creatorcontrib><creatorcontrib>Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)</creatorcontrib><title>Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering</title><title>Nucleic acids research</title><addtitle>Nucleic Acids Res</addtitle><description>Abstract
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.</description><subject>Amino Acid Motifs</subject><subject>Amino Acid Sequence</subject><subject>BASIC BIOLOGICAL SCIENCES</subject><subject>Circoviridae - enzymology</subject><subject>Conserved Sequence</subject><subject>Crystallography, X-Ray</subject><subject>Deoxyribonuclease I - chemistry</subject><subject>Deoxyribonuclease I - metabolism</subject><subject>DNA Helicases - chemistry</subject><subject>DNA Helicases - metabolism</subject><subject>DNA, Single-Stranded - chemistry</subject><subject>DNA, Single-Stranded - metabolism</subject><subject>Gene Library</subject><subject>Massively Parallel (Deep) Sequencing</subject><subject>Models, Molecular</subject><subject>Molecular Docking Simulation</subject><subject>Molecular Sequence Data</subject><subject>Nucleic Acid Conformation</subject><subject>Nucleic Acid Enzymes</subject><subject>Nucleic Acid Enzymology</subject><subject>Plant Viruses - enzymology</subject><subject>Protein Binding</subject><subject>Protein Conformation</subject><subject>Protein Engineering - methods</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Replication Origin</subject><subject>Sequence Alignment</subject><subject>Sequence Homology, Amino Acid</subject><subject>Single-Strand Specific DNA and RNA Endonucleases - chemistry</subject><subject>Single-Strand Specific DNA and RNA Endonucleases - metabolism</subject><subject>Substrate Specificity</subject><subject>Trans-Activators - chemistry</subject><subject>Trans-Activators - metabolism</subject><subject>Viral Proteins - chemistry</subject><subject>Viral Proteins - metabolism</subject><issn>0305-1048</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>TOX</sourceid><sourceid>EIF</sourceid><recordid>eNp9kU1v1DAQhi1ERZeFG2dkcaEHQj1OnI8LUtVCF6mAhOjZcpxJanDs1E6q7om_jtvdVnDhZFt-5pnRvIS8AvYeWJMfOxWOh19KAS_qJ2QFecmzoin5U7JiORMZsKI-JM9j_MkYFCCKZ-Qwz4tUC7Aiv794i3qxKtDFdRgm45xxQ6S-pzGefT2hcUJteqPNvKXtln7HiW4uNxm6zrtFW1QRI1Wuo2acrNFqNt5F2vtwj81qoONiZzNZvE3iexLdYBxiSO8X5KBXNuLL_bkml58-_jjdZBffzj-fnlxkWrBmzpAVvO51CwLyru5VyfK2KVooa1ZXjYKK1UKItuElVx1opWuerrrkDWCeavM1-bDzTks7YqfRzUFZOQUzqrCVXhn5748zV3LwN7Kqkyi1W5M3O4GPs5ExrQP1lfbOoZ4lVCJts0nQ0b5L8NcLxlmOJmq0Vjn0S5S8qErgAuDO926H6uBjDNg_zgJM3gUrU7DyIdiEv_57_kf4IckEvN3Pt0z_V_0BfOeuYQ</recordid><startdate>20210125</startdate><enddate>20210125</enddate><creator>Tompkins, Kassidy J</creator><creator>Houtti, Mo</creator><creator>Litzau, Lauren A</creator><creator>Aird, Eric J</creator><creator>Everett, Blake A</creator><creator>Nelson, Andrew T</creator><creator>Pornschloegl, Leland</creator><creator>Limón-Swanson, Lidia K</creator><creator>Evans, Robert L</creator><creator>Evans, Karen</creator><creator>Shi, Ke</creator><creator>Aihara, Hideki</creator><creator>Gordon, Wendy R</creator><general>Oxford University Press</general><scope>TOX</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>OTOTI</scope><scope>5PM</scope></search><sort><creationdate>20210125</creationdate><title>Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering</title><author>Tompkins, Kassidy J ; Houtti, Mo ; Litzau, Lauren A ; Aird, Eric J ; Everett, Blake A ; Nelson, Andrew T ; Pornschloegl, Leland ; Limón-Swanson, Lidia K ; Evans, Robert L ; Evans, Karen ; Shi, Ke ; Aihara, Hideki ; Gordon, Wendy R</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c509t-e0428fcb1513d8fa603b94b1680879a1708555b9262ad1cac82262c6291e30423</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2021</creationdate><topic>Amino Acid Motifs</topic><topic>Amino Acid Sequence</topic><topic>BASIC BIOLOGICAL SCIENCES</topic><topic>Circoviridae - enzymology</topic><topic>Conserved Sequence</topic><topic>Crystallography, X-Ray</topic><topic>Deoxyribonuclease I - chemistry</topic><topic>Deoxyribonuclease I - metabolism</topic><topic>DNA Helicases - chemistry</topic><topic>DNA Helicases - metabolism</topic><topic>DNA, Single-Stranded - chemistry</topic><topic>DNA, Single-Stranded - metabolism</topic><topic>Gene Library</topic><topic>Massively Parallel (Deep) Sequencing</topic><topic>Models, Molecular</topic><topic>Molecular Docking Simulation</topic><topic>Molecular Sequence Data</topic><topic>Nucleic Acid Conformation</topic><topic>Nucleic Acid Enzymes</topic><topic>Nucleic Acid Enzymology</topic><topic>Plant Viruses - enzymology</topic><topic>Protein Binding</topic><topic>Protein Conformation</topic><topic>Protein Engineering - methods</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Replication Origin</topic><topic>Sequence Alignment</topic><topic>Sequence Homology, Amino Acid</topic><topic>Single-Strand Specific DNA and RNA Endonucleases - chemistry</topic><topic>Single-Strand Specific DNA and RNA Endonucleases - metabolism</topic><topic>Substrate Specificity</topic><topic>Trans-Activators - chemistry</topic><topic>Trans-Activators - metabolism</topic><topic>Viral Proteins - chemistry</topic><topic>Viral Proteins - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Tompkins, Kassidy J</creatorcontrib><creatorcontrib>Houtti, Mo</creatorcontrib><creatorcontrib>Litzau, Lauren A</creatorcontrib><creatorcontrib>Aird, Eric J</creatorcontrib><creatorcontrib>Everett, Blake A</creatorcontrib><creatorcontrib>Nelson, Andrew T</creatorcontrib><creatorcontrib>Pornschloegl, Leland</creatorcontrib><creatorcontrib>Limón-Swanson, Lidia K</creatorcontrib><creatorcontrib>Evans, Robert L</creatorcontrib><creatorcontrib>Evans, Karen</creatorcontrib><creatorcontrib>Shi, Ke</creatorcontrib><creatorcontrib>Aihara, Hideki</creatorcontrib><creatorcontrib>Gordon, Wendy R</creatorcontrib><creatorcontrib>Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)</creatorcontrib><collection>Oxford Journals Open Access Collection</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>OSTI.GOV</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nucleic acids research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Tompkins, Kassidy J</au><au>Houtti, Mo</au><au>Litzau, Lauren A</au><au>Aird, Eric J</au><au>Everett, Blake A</au><au>Nelson, Andrew T</au><au>Pornschloegl, Leland</au><au>Limón-Swanson, Lidia K</au><au>Evans, Robert L</au><au>Evans, Karen</au><au>Shi, Ke</au><au>Aihara, Hideki</au><au>Gordon, Wendy R</au><aucorp>Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)</aucorp><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucleic Acids Res</addtitle><date>2021-01-25</date><risdate>2021</risdate><volume>49</volume><issue>2</issue><spage>1046</spage><epage>1064</epage><pages>1046-1064</pages><issn>0305-1048</issn><eissn>1362-4962</eissn><abstract>Abstract
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.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>33410911</pmid><doi>10.1093/nar/gkaa1248</doi><tpages>19</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Amino Acid Motifs Amino Acid Sequence BASIC BIOLOGICAL SCIENCES Circoviridae - enzymology Conserved Sequence Crystallography, X-Ray Deoxyribonuclease I - chemistry Deoxyribonuclease I - metabolism DNA Helicases - chemistry DNA Helicases - metabolism DNA, Single-Stranded - chemistry DNA, Single-Stranded - metabolism Gene Library Massively Parallel (Deep) Sequencing Models, Molecular Molecular Docking Simulation Molecular Sequence Data Nucleic Acid Conformation Nucleic Acid Enzymes Nucleic Acid Enzymology Plant Viruses - enzymology Protein Binding Protein Conformation Protein Engineering - methods Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - metabolism Replication Origin Sequence Alignment Sequence Homology, Amino Acid Single-Strand Specific DNA and RNA Endonucleases - chemistry Single-Strand Specific DNA and RNA Endonucleases - metabolism Substrate Specificity Trans-Activators - chemistry Trans-Activators - metabolism Viral Proteins - chemistry Viral Proteins - metabolism |
| title | Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering |
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