Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7
KAUST DepartmentBiological and Environmental Sciences and Engineering (BESE) Division
Laboratory of DNA Replication and Recombination
Online Publication Date2010-03-28
Print Publication Date2010-07-01
Permanent link to this recordhttp://hdl.handle.net/10754/325449
MetadataShow full item record
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.
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.
PublisherOxford University Press (OUP)
JournalNucleic Acids Research
PubMed Central IDPMC2910064
The following license files are associated with this item:
Except where otherwise noted, this item's license is described as This 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.
- Interaction of ribonucleoside triphosphates with the gene 4 primase of bacteriophage T7.
- Authors: Frick DN, Kumar S, Richardson CC
- Issue date: 1999 Dec 10
- The roles of tryptophans in primer synthesis by the DNA primase of bacteriophage T7.
- Authors: Zhang H, Lee SJ, Richardson CC
- Issue date: 2012 Jul 6
- Interaction of bacteriophage T7 gene 4 primase with its template recognition site.
- Authors: Frick DN, Richardson CC
- Issue date: 1999 Dec 10
- Acidic residues in the nucleotide-binding site of the bacteriophage T7 DNA primase.
- Authors: Lee SJ, Richardson CC
- Issue date: 2005 Jul 22
- Lagging strand synthesis in coordinated DNA synthesis by bacteriophage t7 replication proteins.
- Authors: Lee J, Chastain PD 2nd, Griffith JD, Richardson CC
- Issue date: 2002 Feb 8