Combined Solution and Crystal Methods Reveal the Electrostatic Tethers That Provide a Flexible Platform for Replication Activities in the Bacteriophage T7 Replisome

Abstract

Recent structural studies of the bacteriophage T7 DNA replication system have shed light on how multiple proteins assemble to copy two antiparallel DNA strands. In T7, acidic C-terminal tails of both the primase-helicase and single-stranded DNA binding protein bind to two basic patches on the DNA polymerase to aid in replisome assembly, processivity, and coordinated DNA synthesis. Although these electrostatic interactions are essential for DNA replication, the molecular details for how these tails bind the polymerase are unknown. We have determined an X-ray crystal structure of the T7 DNA polymerase bound to both a primer/template DNA and a peptide that mimics the C-terminal tail of the primase-helicase. The structure reveals that the essential C-terminal phenylalanine of the tail binds to a hydrophobic pocket that is surrounded by positive charge on the surface of the polymerase. We show that alterations of polymerase residues that engage the tail lead to defects in viral replication. In the structure, we also observe dTTP bound in the exonuclease active site and stacked against tryptophan 160. Using both primer/extension assays and high-throughput sequencing, we show how mutations in the exonuclease active site lead to defects in mismatch repair and an increase in the level of mutagenesis of the T7 genome. Finally, using small-angle X-ray scattering, we provide the first solution structures of a complex between the single-stranded DNA binding protein and the DNA polymerase and show how a single-stranded DNA binding protein dimer engages both one and two copies of DNA polymerase.

Structural Characterization of Sphingomonas sp. KT-1 PahZ1-Catalyzed Biodegradation of Thermally Synthesized Poly(aspartic acid)

“…The scattering vector is defined as q = 4π sin θ/λ, where 2θ is the scattering angle. All experiments were performed at 20 °C and data was collected and processed as previously described . A SAXS flow cell was directly coupled with an online Agilent 1260 Infinity HPLC system using a Shodex KW803 column.…”

“…All experiments were performed at 20 °C and data was collected and processed as previously described. 24 A SAXS flow cell was directly coupled with an online Agilent 1260 Infinity HPLC system using a Shodex KW803 column. The column was equilibrated with a running buffer (20 mM Tris pH 7.0, 100 mM NaCl, 1 mM DTT) with a flow rate of 0.6 mL/min.…”

Conformational dynamics during high-fidelity DNA replication and translocation defined using a DNA polymerase with a fluorescent artificial amino acid

“…Magnesium ions are shown as green spheres in the exonuclease and polymerase active sites. The structure was drawn using Pymol from PDBID: 6p7e ( 41 ). …”

Correlated Target Search by Vaccinia Virus Uracil–DNA Glycosylase, a DNA Repair Enzyme and a Processivity Factor of Viral Replication Machinery

“…Other than vvUNG, the only known DNA polymerase processivity factor not belonging to the clamp superfamily is thioredoxin, a bacterial protein adopted as a processivity subunit by bacteriophage T7 DNA polymerase. Thioredoxin, however, does not seem to contact DNA directly but rather stabilizes a long polymerase loop that tracks along DNA [69][70][71][72]. Additionally, single-strand binding proteins, although not considered processivity subunits in a strict sense, often enhance the processivity of DNA polymerases and have been used toward this end in fusion constructs [73,74].…”