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

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

Deadline for manuscript submissions: 31 January 2022.

Special Issue Editor

Dr. Modesto Redrejo Rodriguez
E-Mail Website
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

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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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Article
Structural Studies of HNA Substrate Specificity in Mutants of an Archaeal DNA Polymerase Obtained by Directed Evolution
Biomolecules 2020, 10(12), 1647; https://doi.org/10.3390/biom10121647 - 08 Dec 2020
Cited by 3 | Viewed by 1267
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 [...] Read more.
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
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Article
Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom
Biomolecules 2020, 10(2), 203; https://doi.org/10.3390/biom10020203 - 30 Jan 2020
Cited by 1 | Viewed by 1049
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 [...] Read more.
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
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Article
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(11), 648; https://doi.org/10.3390/biom9110648 - 24 Oct 2019
Cited by 2 | Viewed by 1313
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 [...] Read more.
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
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Review

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Review
Composition and Function of Telomerase—A Polymerase Associated with the Origin of Eukaryotes
Biomolecules 2020, 10(10), 1425; https://doi.org/10.3390/biom10101425 - 08 Oct 2020
Cited by 6 | Viewed by 1190
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 [...] Read more.
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
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