Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7

Handle URI:
http://hdl.handle.net/10754/325449
Title:
Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7
Authors:
Lee, Seung-Joo; Zhu, Bin; Hamdan, Samir; Richardson, Charles C.
Abstract:
DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5'-TGGTC-3') than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain. The Author(s) 2010. Published by Oxford University Press.
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division
Citation:
Lee S-J, Zhu B, Hamdan SM, Richardson CC (2010) Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7. Nucleic Acids Research 38: 4372-4383. doi:10.1093/nar/gkq205.
Publisher:
Oxford University Press
Journal:
Nucleic Acids Research
Issue Date:
28-Mar-2010
DOI:
10.1093/nar/gkq205
PubMed ID:
20350931
PubMed Central ID:
PMC2910064
Type:
Article
ISSN:
03051048
Appears in Collections:
Articles; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.authorLee, Seung-Jooen
dc.contributor.authorZhu, Binen
dc.contributor.authorHamdan, Samiren
dc.contributor.authorRichardson, Charles C.en
dc.date.accessioned2014-08-27T09:51:49Z-
dc.date.available2014-08-27T09:51:49Z-
dc.date.issued2010-03-28en
dc.identifier.citationLee S-J, Zhu B, Hamdan SM, Richardson CC (2010) Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7. Nucleic Acids Research 38: 4372-4383. doi:10.1093/nar/gkq205.en
dc.identifier.issn03051048en
dc.identifier.pmid20350931en
dc.identifier.doi10.1093/nar/gkq205en
dc.identifier.urihttp://hdl.handle.net/10754/325449en
dc.description.abstractDNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5'-TGGTC-3') than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain. The Author(s) 2010. Published by Oxford University Press.en
dc.language.isoenen
dc.publisherOxford University Pressen
dc.rightsThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.en
dc.rights.urihttp://creativecommons.org/licenses/by-nc/2.5en
dc.subjectadenosine triphosphateen
dc.subjectcytidine triphosphateen
dc.subjectDNA primaseen
dc.subjectnucleoside triphosphataseen
dc.subjectbacteriophage T7en
dc.subjectbinding affinityen
dc.subjectbinding siteen
dc.subjectcontrolled studyen
dc.subjectDNA bindingen
dc.subjectDNA determinationen
dc.subjectDNA sequenceen
dc.subjectDNA synthesisen
dc.subjectDNA templateen
dc.subjectgene functionen
dc.subjectsurface plasmon resonanceen
dc.subjectAdenosine Triphosphateen
dc.subjectBacteriophage T7en
dc.subjectBase Sequenceen
dc.subjectBinding Sitesen
dc.subjectCytidine Triphosphateen
dc.subjectDNA Primaseen
dc.subjectDNA, Single-Strandeden
dc.subjectDNA-Binding Proteinsen
dc.subjectModels, Molecularen
dc.subjectOligoribonucleotidesen
dc.subjectProtein Bindingen
dc.subjectProtein Structure, Tertiaryen
dc.subjectTemplates, Geneticen
dc.titleMechanism of sequence-specific template binding by the DNA primase of bacteriophage T7en
dc.typeArticleen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
dc.identifier.journalNucleic Acids Researchen
dc.identifier.pmcidPMC2910064en
dc.eprint.versionPublisher's Version/PDFen
dc.contributor.institutionDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, United Statesen
dc.contributor.affiliationKing Abdullah University of Science and Technology (KAUST)en
kaust.authorHamdan, Samiren

Related articles on PubMed

This item is licensed under a Creative Commons License
Creative Commons
All Items in KAUST are protected by copyright, with all rights reserved, unless otherwise indicated.