Deshpande, R. A. | Search bioRxiv

bioRxiv2025

Continuous hypermutation and evolution of luciferase variants

Several luciferases have been developed for imaging and biosensing, and the collection continues to grow as new applications are pursued. The current workflow for luciferase optimization, while successful, remains laborious and inefficient. Mutant libraries are generated in vitro and screened, "winning" mutants are picked by hand, and the isolated sequences are subjected to additional rounds of mutagenesis and screening. Here, we present a streamlined platform for luciferase engineering that removes the need for manual library generation during each cycle. We purposed an orthogonal DNA replication (OrthoRep) system for continuous hypermutation of a well-known luciferase (GeNL). Short cycles of culturing and screening were sufficient to evolve the enzyme, with no repetitive manual library generation necessary. New GeNL variants were identified that exhibit improved light outputs with a non-cognate and inexpensive luciferin. We further characterized the novel luciferases in cell models. Collectively this work establishes OrthoRep and continuous hypermutation as a viable method to engineer luciferases, and sets the stage for more rapid development of bioluminescent reporters. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/669707v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@3959dborg.highwire.dtl.DTLVardef@13a57c0org.highwire.dtl.DTLVardef@1572c04org.highwire.dtl.DTLVardef@abd59_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice

The Mre11-Rad50-Nbs1 (MRN) complex recognizes and processes DNA double-strand breaks for homologous recombination by performing short-range removal of 5' strands. Endonucleolytic processing by MRN requires a stably bound protein at the break site--a role we postulate is played by DNA-dependent protein kinase (DNA-PK) in mammals. Here we interrogate the sites of MRN-dependent processing by isolating and sequencing DNA-PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated with highest efficiency when DNA-PK is catalytically blocked, yielding products within 200 bp of the break site, whereas DNA-PK products in the absence of kinase inhibition show much greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA-PK and Mre11 binding, showing that Mre11 and DNA-PK both bind to DNA ends before release of DNA-PK-bound products. These results support a sequential model of double-strand break repair involving collaborative interactions between homologous and non-homologous repair complexes.

molecular biology↗