Publication Date




Embargo Period


Degree Type


Degree Name

Doctor of Philosophy (PHD)


Biochemistry and Molecular Biology (Medicine)

Date of Defense


First Committee Member

Paul C. Schiller

Second Committee Member

Pedro J.I. Salas

Third Committee Member

Richard S. Myers

Fourth Committee Member

Ramiro E. Verdun

Fifth Committee Member

Stephan Züchner


Genome editing by homologous recombination (HR) is centrally important for functional genomics, making disease models, bioengineering and for gene therapy. Viral HR relies on a SynExo complex comprised of an exonuclease and a synaptase. Genome editing by the phage lambda synaptase Beta approaches HR rates of 10% in E. coli using ssDNA-mediated Recombineering but is much less efficient in mammalian cells. SynExos are common to dsDNA viruses that infect a variety of hosts and their mechanisms are conserved, suggesting that direct genome Recombineering can be extended beyond bacteria. Our hypothesis is that co-evolution of viral SynExo recombinases with host proteins makes Recombineering host-specific. We predict that human Herpes virus ICP8 protein can catalyze HR in human cells with greater efficiency than Beta protein. To test the hypothesis, we developed a ssDNA Recombineering assay based on fluorescent protein engineering in vivo, created several novel fluorescent proteins, and optimized parameters to increase gene editing rates in bacteria to ~40% of transformed cells. The Recombineering assay was subsequently moved into human cells expressing ICP8 or “humanized” Beta (HumBeta). Optimized Recombineering in human cells was stimulated by ICP8 above endogenous gene targeting rates. In contrast, HumBeta did not stimulate gene targeting in human cells despite being properly expressed and localized, indicating that Recombineering is host specific. Evidence for host-specific protein interactions in Recombineering was obtained using proteomics and in vitro functional assays. We propose that viral Recombineering is coordinated with host recombination functions, coupled to replisome recruitment, and terminated by proteases and chaperones.


Recombineering, ICP8, Beta, gene targeting, HHV1, phage, fluorescent proteins