Herpes ICP8 protein stimulates homologous recombination in human cells

Mapping Intimacies ◽

10.1101/259739 ◽

2018 ◽

Author(s):

Melvys Valledor ◽

Richard S. Myers ◽

Paul C. Schiller

Keyword(s):

Homologous Recombination ◽

Gene Targeting ◽

Genomic Analysis ◽

Human Cells ◽

E Coli ◽

Recombination Proteins ◽

Genome Modification ◽

Functional Genomic Analysis ◽

Human Herpes Virus Type

ABSTRACTRecombineering has transformed functional genomic analysis. Genome modification by recombineering using the phage lambda Red SynExo homologous recombination proteins Beta inEscherichia colihas approached 100% efficiency. While highly efficient inE. coli, recombineering using the Red SynExo in other organisms declines in efficiency roughly correlating with phylogenetic distance fromE. coli. SynExo recombinases are common to double-stranded DNA viruses infecting a variety of organisms, including humans. Human Herpes virus Type 1 (HHV1) encodes a SynExo comprised of ICP8 synaptase and UL12 exonuclease. In a previous study, the Herpes SynExo was reconstitutedin vitroand shown to catalyze a model recombination reaction. Here we describe stimulation of gene targeting to edit a novel fluorescent protein gene in the human genome using ICP8 and compared its efficiency to that of a “humanized” version of Beta protein from phage λ. ICP8 significantly enhanced gene targeting rates in HEK 293 T cells while Beta was not only unable to catalyze recombineering but inhibited gene targeting using endogenous recombination functions, despite both synaptases being well-expressed and localized to the nucleus. This proof of concept encourages developing species-specific SynExo recombinases for genome engineering.SIGNIFICANCEGenome modification by recombineering using SynExo viral recombination proteins has transformed functional genomic analysis in bacteria. Single-stranded DNA (ssDNA) recombineering approaches 100% efficiency inE. coliusing Beta protein from bacteriophage lambda, but recombineering has not been extended to eukaryotic genomes. Efficient recombineering requires SynExos that co-evolved with a viral host, however SynExos are common to viruses infecting a variety of organisms, including humans. The ICP8 protein of Human Herpes virus Type 1 is a SynExo protein similar to Beta. In this pioneering study, Herpes ICP8 stimulated gene targeting in a human genome by homologous recombination while the bacterial virus Beta protein inhibited recombination in human cells. This is the first demonstration of host-specific recombineering in human cells using a human viral SynExo protein.

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Faculty Opinions recommendation of Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1005245.61705 ◽

2002 ◽

Author(s):

Lisa Denzin

Keyword(s):

Genomic Analysis ◽

Functional Genomic ◽

E Coli ◽

Functional Genomic Analysis

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EXO1 Plays a Role in Generating Type I and Type II Survivors in Budding Yeast

Genetics ◽

10.1093/genetics/166.4.1641 ◽

2004 ◽

Vol 166 (4) ◽

pp. 1641-1649

Author(s):

Laura Maringele ◽

David Lydall

Keyword(s):

Homologous Recombination ◽

Budding Yeast ◽

Human Cells ◽

Telomere Maintenance ◽

Recombination Process ◽

Yeast Cells ◽

Type I ◽

Type Ii ◽

Recombination Proteins

Abstract Telomerase-defective budding yeast cells escape senescence by using homologous recombination to amplify telomeric or subtelomeric structures. Similarly, human cells that enter senescence can use homologous recombination for telomere maintenance, when telomerase cannot be activated. Although recombination proteins required to generate telomerase-independent survivors have been intensively studied, little is known about the nucleases that generate the substrates for recombination. Here we demonstrate that the Exo1 exonuclease is an initiator of the recombination process that allows cells to escape senescence and become immortal in the absence of telomerase. We show that EXO1 is important for generating type I survivors in yku70Δ mre11Δ cells and type II survivors in tlc1Δ cells. Moreover, in tlc1Δ cells, EXO1 seems to contribute to the senescence process itself.

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Application of Functional Genomics to Pathway Optimization for Increased Isoprenoid Production

Applied and Environmental Microbiology ◽

10.1128/aem.02750-07 ◽

2008 ◽

Vol 74 (10) ◽

pp. 3229-3241 ◽

Cited By ~ 127

Author(s):

Lance Kizer ◽

Douglas J. Pitera ◽

Brian F. Pfleger ◽

Jay D. Keasling

Keyword(s):

Biosynthetic Pathway ◽

Fatty Acid Biosynthesis ◽

Cell Metabolism ◽

Genomic Analysis ◽

Acid Biosynthesis ◽

Cytotoxic Effects ◽

E Coli ◽

Pathway Optimization ◽

Functional Genomic Analysis ◽

Microbial Host

ABSTRACT Producing complex chemicals using synthetic metabolic pathways in microbial hosts can have many advantages over chemical synthesis but is often complicated by deleterious interactions between pathway intermediates and the host cell metabolism. With the maturation of functional genomic analysis, it is now technically feasible to identify modes of toxicity associated with the accumulation of foreign molecules in the engineered bacterium. Previously, Escherichia coli was engineered to produce large quantities of isoprenoids by creating a mevalonate-based isopentenyl pyrophosphate biosynthetic pathway (V. J. J. Martin et al., Nat. Biotechnol. 21:796-802, 2003). The engineered E. coli strain produced high levels of isoprenoids, but further optimization led to an imbalance in carbon flux and the accumulation of the pathway intermediate 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA), which proved to be cytotoxic to E. coli. Using both DNA microarray analysis and targeted metabolite profiling, we have studied E. coli strains inhibited by the intracellular accumulation of HMG-CoA. Our results indicate that HMG-CoA inhibits fatty acid biosynthesis in the microbial host, leading to generalized membrane stress. The cytotoxic effects of HMG-CoA accumulation can be counteracted by the addition of palmitic acid (16:0) and, to a lesser extent, oleic acid (cis-Δ9-18:1) in the growth medium. This work demonstrates the utility of using transcriptomic and metabolomic methods to optimize synthetic biological systems.

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