Structures of an Apo and a Binary Complex of an Evolved Archeal B Family DNA Polymerase Capable of Synthesising Highly Cy-Dye Labelled DNA

Wynne, Samantha A.; Pinheiro, Vitor B.; Holliger, Philipp; Leslie, Andrew G. W.

doi:10.1371/journal.pone.0070892

Cited by 31 publications

(25 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Based on these observations and available cocrystal structures of related Pfu and KOD polymerases in complex with their DNA substrates [28,29], we proposed a model for the putative priming complex. The crystallographic evidence indicates that the polymerases tightly bind the duplex part of the DNA, whereas the 5 0 part of the template strand is less structured and can assume multiple conformations (S1 Fig).…”

Section: Plos Biologymentioning

confidence: 98%

Precise genomic mapping of 5-hydroxymethylcytosine via covalent tether-directed sequencing

et al. 2020

View full text Add to dashboard Cite

5-hydroxymethylcytosine (5hmC) is the most prevalent intermediate on the oxidative DNA demethylation pathway and is implicated in regulation of embryogenesis, neurological processes, and cancerogenesis. Profiling of this relatively scarce genomic modification in clinical samples requires cost-effective high-resolution techniques that avoid harsh chemical treatment. Here, we present a bisulfite-free approach for 5hmC profiling at single-nucleotide resolution, named hmTOP-seq (5hmC-specific tethered oligonucleotide-primed sequencing), which is based on direct sequence readout primed at covalently labeled 5hmC sites from an in situ tethered DNA oligonucleotide. Examination of distinct conjugation chemistries suggested a structural model for the tether-directed nonhomologous polymerase priming enabling theoretical evaluation of suitable tethers at the design stage. The hmTOP-seq procedure was optimized and validated on a small model genome and mouse embryonic stem cells, which allowed construction of single-nucleotide 5hmC maps reflecting subtle differences in strand-specific CG hydroxymethylation. Collectively, hmTOP-seq provides a new valuable tool for cost-effective and precise identification of 5hmC in characterizing its biological role and epigenetic changes associated with human disease.

show abstract

Section: Plos Biologymentioning

confidence: 98%

Precise genomic mapping of 5-hydroxymethylcytosine via covalent tether-directed sequencing

et al. 2020

View full text Add to dashboard Cite

show abstract

“…5, 20 The 4AIL structure captured PolB in polymerization mode. The binary structures were superimposed on the thumb domain of PolB (residues D636 to G750).…”

Section: Methodsmentioning

confidence: 99%

“…4–5, 9–12 In PolB the switch from pol to exo activity is accompanied by large conformational changes of both the polymerase and DNA, as evident from crystal structures of PolB in the replication and editing modes. While structures of archaeal PolB/DNA complexes in pol and exo mode exist, details of the switching mechanism have remained elusive.…”

Section: Introductionmentioning

confidence: 99%

Secondary Interaction Interfaces with PCNA Control Conformational Switching of DNA Polymerase PolB from Polymerization to Editing

Cheng

Kossmann

et al. 2016

J. Phys. Chem. B

View full text Add to dashboard Cite

Replicative DNA polymerases (Pols) frequently possess two distinct DNA processing activities – DNA synthesis (polymerization) and proofreading (3′–5′ exonuclease activity). The polymerase and exonuclease reactions are performed alternately and are spatially separated in different protein domains. Thus, the growing DNA primer terminus has to undergo dynamic conformational switching between two distinct functional sites on the polymerase. Furthermore, the transition from polymerization (pol) to exonuclease (exo) mode must occur in the context of a DNA Pol holoenzyme, wherein the polymerase is physically associated with the processivity factor Proliferating Cell Nuclear Antigen (PCNA) and primer-template DNA. The mechanism of this conformational switching and the role that PCNA plays in it had remained obscure, largely due to the dynamic nature of the ternary Pol/PCNA/DNA assemblies. Here, we present computational models of the ternary assemblies for the archaeal polymerase PolB. We have combined all available structural information for the binary complexes with electron microscopy (EM) data and have refined atomistic models for the ternary PolB/PCNA/DNA assemblies in the pol and exo modes using molecular dynamics simulations. In addition to the canonical PIP-box/inter-domain connector loop (IDCL) interface of PolB with PCNA, contact analysis of the simulation trajectories revealed new secondary binding interfaces, distinct between the pol and exo states. Using targeted molecular dynamics (TMD), we explored the conformational transition from pol to exo mode. We identified a hinge region between the thumb and palm domain of PolB that is critical for conformational switching. With the thumb domain anchored onto the PCNA surface, the neighboring palm domain executed rotational motion around the hinge, bringing the core of PolB down toward PCNA to form a new interface with the clamp. A helix from PolB containing a patch of arginine residues was involved in the binding, locking the complex in the exo-mode conformation. Together, these results provide a structural view of how the transition between the pol and exo states of PolB is coordinated through PCNA to achieve efficient proofreading.

show abstract

“…Construction of a simple model of the complex that Pcpolymerase forms with DNA was undertaken by fitting our structure of the enzyme to the DNA-bound structure of P. furiosus DNA polymerase (PDB entry 4ail; Wynne et al, 2013). This structure was derived from a mutant form of Pfu which was co-crystallized with a primer-template duplex of DNA with a 2 0 -3 0 dideoxy terminal cytosine at the 3 0 end of the primer strand to stall the polymerase.…”

Section: Model Of the Complex With Dnamentioning

confidence: 99%

Structure of the family B DNA polymerase from the hyperthermophilic archaeonPyrobaculum calidifontis

Guo¹,

Zhang²,

Coker³

et al. 2017

Acta Cryst Sect D Struct Biol

View full text Add to dashboard Cite

The family B DNA polymerase from Pyrobaculum calidifontis (Pc-polymerase) consists of 783 amino acids and is magnesium-ion dependent. It has an optimal pH of 8.5, an optimal temperature of 75 C and a half-life of 4.5 h at 95 C, giving it greater thermostability than the widely used Taq DNA polymerase. The enzyme is also capable of PCR-amplifying larger DNA fragments of up to 7.5 kb in length. It was shown to have functional, error-correcting 3 0 -5 0 exonuclease activity, as do the related high-fidelity DNA polymerases from Pyrococcus furiosus, Thermococcus kodakarensis KOD1 and Thermococcus gorgonarius, which have extensive commercial applications. Pc-polymerase has a quite low sequence identity of approximately 37% to these enzymes, which, in contrast, have very high sequence identity to each other, suggesting that the P. calidifontis enzyme is distinct. Here, the structure determination of Pc-polymerase is reported, which has been refined to an R factor of 24.47% and an R free of 28.81% at 2.80 Å resolution. The domains of the enzyme are arranged in a circular fashion to form a disc with a narrow central channel. One face of the disc has a number of connected crevices in it, which allow the protein to bind duplex and single-stranded DNA. The central channel is thought to allow incoming nucleoside triphosphates to access the active site. The enzyme has a number of unique structural features which distinguish it from other archaeal DNA polymerases and may account for its high processivity. A model of the complex with the primer-template duplex of DNA indicates that the largest conformational change that occurs upon DNA binding is the movement of the thumb domain, which rotates by 7.6 and moves by 10.0 Å . The surface potential of the enzyme is dominated by acidic groups in the central region of the molecule, where catalytic magnesium ions bind at the polymerase and exonuclease active sites. The outer regions are richer in basic amino acids that presumably interact with the sugar-phosphate backbone of DNA. The large number of salt bridges may contribute to the high thermal stability of this enzyme.

show abstract

Structures of an Apo and a Binary Complex of an Evolved Archeal B Family DNA Polymerase Capable of Synthesising Highly Cy-Dye Labelled DNA

Cited by 31 publications

References 42 publications

Precise genomic mapping of 5-hydroxymethylcytosine via covalent tether-directed sequencing

Precise genomic mapping of 5-hydroxymethylcytosine via covalent tether-directed sequencing

Secondary Interaction Interfaces with PCNA Control Conformational Switching of DNA Polymerase PolB from Polymerization to Editing

Structure of the family B DNA polymerase from the hyperthermophilic archaeonPyrobaculum calidifontis

Contact Info

Product

Resources

About