Timothy Newing - Academia.edu (original) (raw)

Papers by Timothy Newing

Research paper thumbnail of Genomic multidisciplinary teams: A model for navigating genetic mainstreaming and precision medicine

Journal of paediatrics and child health, Apr 11, 2024

Research paper thumbnail of Half a century after their discovery: Structural insights into exonuclease and annealase proteins catalyzing recombineering

Engineering Microbiology, Feb 29, 2024

Research paper thumbnail of Structure of phage lambda Red-beta(177) annealase shows how it anneals DNA strands during single-strand annealing homologous DNA recombination

bioRxiv (Cold Spring Harbor Laboratory), Apr 9, 2022

Single-strand annealing homologous DNA recombination is a process that is conserved throughout ev... more Single-strand annealing homologous DNA recombination is a process that is conserved throughout evolution from bacteriophages to humans, highlighting its importance and indispensability. It is a process that repairs double-stranded DNA breaks and is particularly vital in dsDNA viruses. The Redβ protein from the bacteriophage lambda is the archetypal annealase protein, forming the basis of our knowledge about this class of proteins. Along with the exonuclease λExo, these two proteins not only form the model system for single-strand annealing homologous recombination, but are also used in thousands of laboratories worldwide for performing genetic manipulations. After its discovery in 1966, we report the first structure of the DNA-binding and oligomerization domain of Redβ, providing details about the mechanism of homologous DNA annealing.

Research paper thumbnail of Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD

Nature Communications, Dec 18, 2020

In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for th... more In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. HelD has a 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria.

Research paper thumbnail of Abstract 2615: Cryo-EM investigation of a single strand annealing homologous DNA recombination protein, ICP8 annealase from HSV-1

Journal of Biological Chemistry, 2023

Research paper thumbnail of RedBeta177 oligomeric helical assembly bound to two complementary 27mer ssDNA oligonucleotides

Research paper thumbnail of Redβ177 annealase structure reveals details of oligomerization and λ Red-mediated homologous DNA recombination

Nature Communications

The Redβ protein of the bacteriophage λ red recombination system is a model annealase which catal... more The Redβ protein of the bacteriophage λ red recombination system is a model annealase which catalyzes single-strand annealing homologous DNA recombination. Here we present the structure of a helical oligomeric annealing intermediate of Redβ, consisting of N-terminal residues 1-177 bound to two complementary 27mer oligonucleotides, determined via cryogenic electron microscopy (cryo-EM) to a final resolution of 3.3 Å. The structure reveals a continuous binding groove which positions and stabilizes complementary DNA strands in a planar orientation to facilitate base pairing via a network of hydrogen bonding. Definition of the inter-subunit interface provides a structural basis for the propensity of Redβ to oligomerize into functionally significant long helical filaments, a trait shared by most annealases. Our cryo-EM structure and molecular dynamics simulations suggest that residues 133-138 form a flexible loop which modulates access to the binding groove. More than half a century afte...

Research paper thumbnail of Structure of phage λ Redβ177 annealase shows how it anneals DNA strands during single-strand annealing homologous DNA recombination

The bacteriophage λ red recombination system catalyzes the single-strand annealing homologous DNA... more The bacteriophage λ red recombination system catalyzes the single-strand annealing homologous DNA recombination reaction, in which Redβ annealase protein plays a critical role. Using cryogenic electron microscopy, we were able to determine a structure of a C-terminally truncated Redβ with the residues 1-177 bound to two complementary 27mer oligonucleotides forming an annealing intermediate, to a final resolution of 3.3 Å. This structure validates and rationalizes decades of experimental observations on the biochemistry of Redβ. Definition of the interaction surfaces between subunits explains not only the DNA binding properties of Redβ, but also its propensity to oligomerize into long helical filaments, which are also formed by almost all annealases and are known to be functionally important. In addition, this annealing intermediate structure provides a detailed picture of the hydrogen bonding network that positions the DNA strands in a planar orientation to facilitate base pairing. ...

Research paper thumbnail of Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD

Nature Communications

In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for th... more In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. HelD has a 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria.

Research paper thumbnail of Genomic multidisciplinary teams: A model for navigating genetic mainstreaming and precision medicine

Journal of paediatrics and child health, Apr 11, 2024

Research paper thumbnail of Half a century after their discovery: Structural insights into exonuclease and annealase proteins catalyzing recombineering

Engineering Microbiology, Feb 29, 2024

Research paper thumbnail of Structure of phage lambda Red-beta(177) annealase shows how it anneals DNA strands during single-strand annealing homologous DNA recombination

bioRxiv (Cold Spring Harbor Laboratory), Apr 9, 2022

Single-strand annealing homologous DNA recombination is a process that is conserved throughout ev... more Single-strand annealing homologous DNA recombination is a process that is conserved throughout evolution from bacteriophages to humans, highlighting its importance and indispensability. It is a process that repairs double-stranded DNA breaks and is particularly vital in dsDNA viruses. The Redβ protein from the bacteriophage lambda is the archetypal annealase protein, forming the basis of our knowledge about this class of proteins. Along with the exonuclease λExo, these two proteins not only form the model system for single-strand annealing homologous recombination, but are also used in thousands of laboratories worldwide for performing genetic manipulations. After its discovery in 1966, we report the first structure of the DNA-binding and oligomerization domain of Redβ, providing details about the mechanism of homologous DNA annealing.

Research paper thumbnail of Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD

Nature Communications, Dec 18, 2020

In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for th... more In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. HelD has a 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria.

Research paper thumbnail of Abstract 2615: Cryo-EM investigation of a single strand annealing homologous DNA recombination protein, ICP8 annealase from HSV-1

Journal of Biological Chemistry, 2023

Research paper thumbnail of RedBeta177 oligomeric helical assembly bound to two complementary 27mer ssDNA oligonucleotides

Research paper thumbnail of Redβ177 annealase structure reveals details of oligomerization and λ Red-mediated homologous DNA recombination

Nature Communications

The Redβ protein of the bacteriophage λ red recombination system is a model annealase which catal... more The Redβ protein of the bacteriophage λ red recombination system is a model annealase which catalyzes single-strand annealing homologous DNA recombination. Here we present the structure of a helical oligomeric annealing intermediate of Redβ, consisting of N-terminal residues 1-177 bound to two complementary 27mer oligonucleotides, determined via cryogenic electron microscopy (cryo-EM) to a final resolution of 3.3 Å. The structure reveals a continuous binding groove which positions and stabilizes complementary DNA strands in a planar orientation to facilitate base pairing via a network of hydrogen bonding. Definition of the inter-subunit interface provides a structural basis for the propensity of Redβ to oligomerize into functionally significant long helical filaments, a trait shared by most annealases. Our cryo-EM structure and molecular dynamics simulations suggest that residues 133-138 form a flexible loop which modulates access to the binding groove. More than half a century afte...

Research paper thumbnail of Structure of phage λ Redβ177 annealase shows how it anneals DNA strands during single-strand annealing homologous DNA recombination

The bacteriophage λ red recombination system catalyzes the single-strand annealing homologous DNA... more The bacteriophage λ red recombination system catalyzes the single-strand annealing homologous DNA recombination reaction, in which Redβ annealase protein plays a critical role. Using cryogenic electron microscopy, we were able to determine a structure of a C-terminally truncated Redβ with the residues 1-177 bound to two complementary 27mer oligonucleotides forming an annealing intermediate, to a final resolution of 3.3 Å. This structure validates and rationalizes decades of experimental observations on the biochemistry of Redβ. Definition of the interaction surfaces between subunits explains not only the DNA binding properties of Redβ, but also its propensity to oligomerize into long helical filaments, which are also formed by almost all annealases and are known to be functionally important. In addition, this annealing intermediate structure provides a detailed picture of the hydrogen bonding network that positions the DNA strands in a planar orientation to facilitate base pairing. ...

Research paper thumbnail of Molecular basis for RNA polymerase-dependent transcription complex recycling by the helicase-like motor protein HelD

Nature Communications

In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for th... more In bacteria, transcription complexes stalled on DNA represent a major source of roadblocks for the DNA replication machinery that must be removed in order to prevent damaging collisions. Gram-positive bacteria contain a transcription factor HelD that is able to remove and recycle stalled complexes, but it was not known how it performed this function. Here, using single particle cryo-electron microscopy, we have determined the structures of Bacillus subtilis RNA polymerase (RNAP) elongation and HelD complexes, enabling analysis of the conformational changes that occur in RNAP driven by HelD interaction. HelD has a 2-armed structure which penetrates deep into the primary and secondary channels of RNA polymerase. One arm removes nucleic acids from the active site, and the other induces a large conformational change in the primary channel leading to removal and recycling of the stalled polymerase, representing a novel mechanism for recycling transcription complexes in bacteria.