Gene 1.2 protein of bacteriophage T7. Effect on deoxyribonucleotide pools.

Myers, Jill A.; Beauchamp, Benjamin; Richardson, Charles C.

doi:10.1016/s0021-9258(18)61186-x

Cited by 33 publications

(6 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Huber et al found that a Dgt overexpressing optA1 mutant E. coli maintained 50× higher dGTPase concentrations, which lowered dGTP pools 5-fold . Wild-type T7 is able to replicate in these dGTP depleted backgrounds, but foundational studies utilizing optA1 strains do not report impact on EOP. − Here, we again demonstrate that T7 does propagate in a Dgt overexpressing strain but EOP is diminished.…”

Section: Resultsmentioning

confidence: 99%

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield

Brooks,

Morici,

Sandoval

2023

ACS Synth. Biol.

View full text Add to dashboard Cite

Phage therapy to treat life-threatening drugresistant infections has been hampered by technical challenges in phage production. Cell-free bacteriophage synthesis (CFBS) can overcome the limitations of standard phage production methods by manufacturing phage virions in vitro. CFBS mimics intracellular phage assembly using transcription/translation machinery (TXTL) harvested from bacterial lysates and combined with reagents to synthesize proteins encoded by a phage genomic DNA template. These systems may enable rapid phage production and engineering to accelerate phages from bench-to-bedside. TXTL harvested from wild type or commonly used bacterial strains was not optimized for bacteriophage production. Here, we demonstrate that TXTL from genetically modified E. coli BL21 can be used to enhance phage T7 yields in vitro by CFBS. Expression of 18 E. coli BL21 genes was manipulated by inducible CRISPR interference (CRISPRi) mediated by nuclease deficient Cas12a from F. novicida (dFnCas12a) to identify genes implicated in T7 propagation as positive or negative effectors. Genes shown to have a significant effect were overexpressed (positive effectors) or repressed (negative effectors) to modify the genetic background of TXTL harvested for CFBS. Phage T7 CFBS yields were improved by up to 10-fold in vitro through overexpression of translation initiation factor IF-3 (inf C) and small RNAs OxyS and CyaR and by repression of RecC subunit exonuclease RecBCD. Continued improvement of CFBS will mitigate phage manufacturing bottlenecks and lower hurdles to widespread adoption of phage therapy.

show abstract

Section: Resultsmentioning

confidence: 99%

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield

Brooks,

Morici,

Sandoval

2023

ACS Synth. Biol.

View full text Add to dashboard Cite

show abstract

“…7 ). This model addresses longstanding questions about how Gp1.2 can inhibit Dgt in vivo, despite requiring a slow equilibration step that is apparently blocked if substrate binding occurs first ( 8 , 9 ). As described above, GTP is normally in the high micromolar to low millimolar range in cells, so the low micromolar binding constant for GTP implies that Dgt active sites will often be occupied by GTP.…”

Section: Discussionmentioning

confidence: 99%

“…A relevant example of such virus/host interactions is between the E . coli Dgt and the 1.2 gene product (Gp1.2) of bacteriophage T7, a system that was initially discovered and investigated in detail by the Richardson group ( 3 , 8 , 9 ). An E. coli strain expressing elevated levels of Dgt due to an up-mutation in the dgt promoter ( optA1 ) ( 3 ) can no longer be infected by T7 if it is missing a functional Gp1.2 ( 8 ).…”

mentioning

confidence: 99%

“…coli Dgt and that the inability of the mutant 1.2 phage to replicate in this strain was caused by its failure to maintain the necessary dGTP levels for its DNA replication ( 9 ). More precisely, phage T7 breaks down the host DNA following infection to secure an elevated supply of DNA precursors; but the loss of Gp1.2 leads to a 200-fold reduced dGTP level when replicating in the optA1 strain, leading to premature termination of phage DNA replication ( 8 ). Thus, dGTP limitation is a clear mechanism by which E .…”

mentioning

confidence: 99%

See 1 more Smart Citation

Mechanism by which T7 bacteriophage protein Gp1.2 inhibits Escherichia coli dGTPase

Klemm

Singh

Smith

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Levels of the cellular dNTPs, the direct precursors for DNA synthesis, are important for DNA replication fidelity, cell cycle control, and resistance against viruses. Escherichia coli encodes a dGTPase (2′-deoxyguanosine-5′-triphosphate [dGTP] triphosphohydrolase [dGTPase]; dgt gene, Dgt) that establishes the normal dGTP level required for accurate DNA replication but also plays a role in protecting E. coli against bacteriophage T7 infection by limiting the dGTP required for viral DNA replication. T7 counteracts Dgt using an inhibitor, the gene 1.2 product (Gp1.2). This interaction is a useful model system for studying the ongoing evolutionary virus/host “arms race.” We determined the structure of Gp1.2 by NMR spectroscopy and solved high-resolution cryo-electron microscopy structures of the Dgt–Gp1.2 complex also including either dGTP substrate or GTP coinhibitor bound in the active site. These structures reveal the mechanism by which Gp1.2 inhibits Dgt and indicate that Gp1.2 preferentially binds the GTP-bound form of Dgt. Biochemical assays reveal that the two inhibitors use different modes of inhibition and bind to Dgt in combination to yield enhanced inhibition. We thus propose an in vivo inhibition model wherein the Dgt–Gp1.2 complex equilibrates with GTP to fully inactivate Dgt, limiting dGTP hydrolysis and preserving the dGTP pool for viral DNA replication.

show abstract

“…Six years later, Jill Myers and Ben Beauchamp found that dGTP levels in optA1 mutants were extremely low (252,253), leading to the purification of the product of the optA gene, a dGTPase (276). The enzyme is interesting both for its unusual physical properties and for the reaction it catalyzes.…”

Section: Rediscovery Of E Coli Dgtpasementioning

confidence: 99%

Untitled

2015

Annu. Rev. Biochem.

View full text Add to dashboard Cite

I spent my childhood and adolescence in North and South Carolina, attended Duke University, and then entered Duke Medical School. One year in the laboratory of George Schwert in the biochemistry department kindled my interest in biochemistry. After one year of residency on the medical service of Duke Hospital, chaired by Eugene Stead, I joined the group of Arthur Kornberg at Stanford Medical School as a postdoctoral fellow. Two years later I accepted a faculty position at Harvard Medical School, where I remain today. During these 50 years, together with an outstanding group of students, postdoctoral fellows, and collaborators, I have pursued studies on DNA replication. I have experienced the excitement of discovering a number of important enzymes in DNA replication that, in turn, triggered an interest in the dynamics of a replisome. My associations with industry have been stimulating and fostered new friendships. I could not have chosen a better career.

show abstract

Gene 1.2 protein of bacteriophage T7. Effect on deoxyribonucleotide pools.

Cited by 33 publications

References 17 publications

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield

Mechanism by which T7 bacteriophage protein Gp1.2 inhibits Escherichia coli dGTPase

Untitled

Contact Info

Product

Resources

About