Next Article in Journal
Emerging Role of Vitamin D and its Associated Molecules in Pathways Related to Pathogenesis of Thrombosis
Previous Article in Journal
Transcriptomic Analysis Reveals the Roles of Detoxification Systems in Response to Mercury in Chromera velia
Open AccessArticle

The Loop of the TPR1 Subdomain of Phi29 DNA Polymerase Plays a Pivotal Role in Primer-Terminus Stabilization at the Polymerization Active Site

Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain
*
Author to whom correspondence should be addressed.
Contributed equally to this work.
Co-senior authors.
Biomolecules 2019, 9(11), 648; https://doi.org/10.3390/biom9110648
Received: 4 September 2019 / Revised: 21 October 2019 / Accepted: 22 October 2019 / Published: 24 October 2019
Bacteriophage Phi29 DNA polymerase belongs to the protein-primed subgroup of family B DNA polymerases that use a terminal protein (TP) as a primer to initiate genome replication. The resolution of the crystallographic structure showed that it consists of an N-terminal domain with the exonuclease activity and a C-terminal polymerization domain. It also has two subdomains specific of the protein-primed DNA polymerases; the TP Regions 1 (TPR1) that interacts with TP and DNA, and 2 (TPR2), that couples both processivity and strand displacement to the enzyme. The superimposition of the structures of the apo polymerase and the polymerase in the polymerase/TP heterodimer shows that the structural changes are restricted almost to the TPR1 loop (residues 304–314). In order to study the role of this loop in binding the DNA and the TP, we changed the residues Arg306, Arg308, Phe309, Tyr310, and Lys311 into alanine, and also made the deletion mutant Δ6 lacking residues Arg306–Lys311. The results show a defective TP binding capacity in mutants R306A, F309A, Y310A, and Δ6. The additional impaired primer-terminus stabilization at the polymerization active site in mutants Y310A and Δ6 allows us to propose a role for the Phi29 DNA polymerase TPR1 loop in the proper positioning of the DNA and TP-priming 3’-OH termini at the preinsertion site of the polymerase to enable efficient initiation and further elongation steps during Phi29 TP-DNA replication. View Full-Text
Keywords: DNA polymerase; replication; protein-priming; terminal protein DNA polymerase; replication; protein-priming; terminal protein
Show Figures

Figure 1

MDPI and ACS Style

del Prado, A.; Santos, E.; Lázaro, J.M.; Salas, M.; de Vega, M. The Loop of the TPR1 Subdomain of Phi29 DNA Polymerase Plays a Pivotal Role in Primer-Terminus Stabilization at the Polymerization Active Site. Biomolecules 2019, 9, 648.

Show more citation formats Show less citations formats
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop