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DNA Polymerases: From the Maintenance of Genetic Information to Applications in Biotechnology and Biomedicine

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A special issue of Biomolecules (ISSN 2218-273X). This special issue belongs to the section "Cellular Biochemistry".

Deadline for manuscript submissions: closed (31 January 2022) | Viewed by 14881

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Special Issue Editor

Dr. Modesto Redrejo Rodriguez

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Guest Editor

Biochemistry Department and Instituto de Investigaciones Biológicas Alberto Sols, Universidad Autónoma de Madrid, Madrid, Spain
Interests: DNA replication, DNA repair, DNA polymerases, DNA amplification, DNA priming, protein engineering, translesion synthesis, genotoxic agents, DNA virus, transposon

Special Issue Information

Dear Colleagues,

Many applications with fundamental importance in modern molecular biology and biomedicine, including polymerase chain reaction (PCR) and whole genome DNA amplification (WGA), as well as some of the state-of-the-art DNA sequencing technologies, would not be feasible without the advances made in characterizing the DNA polymerases (DNAPs) during the last 60 years. Furthermore, the development of WGA at the single-cell and single-molecule level has contributed to some of the most recent breakthroughs in our knowledge of different complex biological systems—from microbial ecosystems, shedding light into microbial dark matter, to human disease, enhancing the sensitivity to detect genetic variants and mutation profiles of individual cells in a tissue or tumor, as well as changing the paradigms in the early diagnosis of cancer and genetic diseases with non-invasive genetic tests. Furthermore, beyond biotechnology applications, DNAPs are the enzymes responsible for preserving genetic information by replicating and repairing nucleic acid molecules in the cells. In the last years, novel developments and experimental approaches have led to a reevaluation of several accepted mechanisms, and have changed many paradigms in the DNA replication and repair field. This Special Issue will cover new advances on the structure, function, and biological role of DNA polymerases, both well-characterized and new, previously overlooked members with unexpected features, including AEPs, like bacterial LigD or PrimPols.

Dr. Modesto Redrejo Rodriguez
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Biomolecules is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

DNA polymerase
DNA replication
DNA repair
TLS
DNA amplification
MDA
PCR
biotechnology

Published Papers (4 papers)

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Research

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25 pages, 3768 KiB

Open AccessArticle

Structural Studies of HNA Substrate Specificity in Mutants of an Archaeal DNA Polymerase Obtained by Directed Evolution

by Camille Samson, Pierre Legrand, Mustafa Tekpinar, Jef Rozenski, Mikhail Abramov, Philipp Holliger, Vitor B. Pinheiro, Piet Herdewijn and Marc Delarue

Biomolecules 2020, 10(12), 1647; https://doi.org/10.3390/biom10121647 - 8 Dec 2020

Cited by 7 | Viewed by 3333

Abstract

Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants. Full article

(This article belongs to the Special Issue DNA Polymerases: From the Maintenance of Genetic Information to Applications in Biotechnology and Biomedicine)

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19 pages, 4218 KiB

Open AccessFeature PaperArticle

Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom

by Alejandro Sánchez-Salvador and Miguel de Vega

Biomolecules 2020, 10(2), 203; https://doi.org/10.3390/biom10020203 - 30 Jan 2020

Cited by 2 | Viewed by 2647

Abstract

The catalytic active site of the Polymerization Domain (PolDom) of bacterial Ligase D is designed to promote realignments of the primer and template strands and extend mispaired 3′ ends. These features, together with the preferred use of ribonucleotides (NTPs) over deoxynucleotides (dNTPs), allow PolDom to perform efficient double strand break repair by nonhomologous end joining when only a copy of the chromosome is present and the intracellular pool of dNTPs is depleted. Here, we evaluate (i) the role of conserved histidine and serine/threonine residues in NTP insertion, and (ii) the importance in the polymerization reaction of a conserved lysine residue that interacts with the templating nucleotide. To that extent, we have analyzed the biochemical properties of variants at the corresponding His651, Ser768, and Lys606 of Pseudomonas aeruginosa PolDom (Pa-PolDom). The results show that preferential insertion of NMPs is principally due to the histidine that also contributes to the plasticity of the active site to misinsert nucleotides. Additionally, Pa-PolDom Lys606 stabilizes primer dislocations. Finally, we show that the active site of PolDom allows the efficient use of 7,8-dihydro-8-oxo-riboguanosine triphosphate (8oxoGTP) as substrate, a major nucleotide lesion that results from oxidative stress, inserting with the same efficiency both the anti and syn conformations of 8oxoGMP. Full article

(This article belongs to the Special Issue DNA Polymerases: From the Maintenance of Genetic Information to Applications in Biotechnology and Biomedicine)

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16 pages, 3261 KiB

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

by Alicia del Prado, Eugenia Santos, José M. Lázaro, Margarita Salas and Miguel de Vega

Biomolecules 2019, 9(11), 648; https://doi.org/10.3390/biom9110648 - 24 Oct 2019

Cited by 3 | Viewed by 3807

Abstract

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. Full article

(This article belongs to the Special Issue DNA Polymerases: From the Maintenance of Genetic Information to Applications in Biotechnology and Biomedicine)

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Review

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24 pages, 3501 KiB

Open AccessReview

Composition and Function of Telomerase—A Polymerase Associated with the Origin of Eukaryotes

by Petra Procházková Schrumpfová and Jiří Fajkus

Biomolecules 2020, 10(10), 1425; https://doi.org/10.3390/biom10101425 - 8 Oct 2020

Cited by 16 | Viewed by 4415

Abstract

The canonical DNA polymerases involved in the replication of the genome are unable to fully replicate the physical ends of linear chromosomes, called telomeres. Chromosomal termini thus become shortened in each cell cycle. The maintenance of telomeres requires telomerase—a specific RNA-dependent DNA polymerase enzyme complex that carries its own RNA template and adds telomeric repeats to the ends of chromosomes using a reverse transcription mechanism. Both core subunits of telomerase—its catalytic telomerase reverse transcriptase (TERT) subunit and telomerase RNA (TR) component—were identified in quick succession in Tetrahymena more than 30 years ago. Since then, both telomerase subunits have been described in various organisms including yeasts, mammals, birds, reptiles and fish. Despite the fact that telomerase activity in plants was described 25 years ago and the TERT subunit four years later, a genuine plant TR has only recently been identified by our group. In this review, we focus on the structure, composition and function of telomerases. In addition, we discuss the origin and phylogenetic divergence of this unique RNA-dependent DNA polymerase as a witness of early eukaryotic evolution. Specifically, we discuss the latest information regarding the recently discovered TR component in plants, its conservation and its structural features. Full article

(This article belongs to the Special Issue DNA Polymerases: From the Maintenance of Genetic Information to Applications in Biotechnology and Biomedicine)

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