Abstract
Limited experimental tools are available to study the consequences of collisions between DNA-bound molecular machines. Here, we repurpose a catalytically inactivated Cas9 (dCas9) construct as a generic, novel, targetable protein–DNA roadblock for studying mechanisms underlying enzymatic activities on DNA substrates in vitro. We illustrate the broad utility of this tool by demonstrating replication fork arrest by the specifically bound dCas9–guideRNA complex to arrest viral, bacterial and eukaryotic replication forks in vitro.
Highlights
The impediment of the progress of DNA replication machinery on template DNA occupied by proteins is an important case in point
Biotinylated target DNA was immobilized on a streptavidin-coated surface and a solution containing dCas9-dL5 pre-programmed with a complementary guide RNA was introduced (Fig. 1b, Supplementary Table S3)
We found that highly purified dCas9-dL5 alone exhibited binding to 83-mer biotinylated dsDNA in the absence of guide RNA (Fig. 1c), consistent with previous work[17]
Summary
The impediment of the progress of DNA replication machinery on template DNA occupied by proteins is an important case in point. Replisomes encounter three major types of protein barriers: transcription complexes, nucleoid-associated proteins, and recombination filaments[6,7,8] Successful replication across such roadblocks requires the coordinated action of several accessory factors and DNA-repair and dedicated restart proteins. Other approaches have involved the introduction of repeat sequences that enable binding of transcription factors to artificially introduce repressor/ operator arrays, or proteins that polymerize to form nucleoprotein filaments[4,12,13,14] Despite their tremendous utility in studying replication fork arrest, these methods suffer from several disadvantages: since the tandem binding of several roadblock proteins is required for effective stalling of the replication fork, the exact positions of the block are often poorly defined. Nuclease dead Cas[9] blocks the progress of replication forks from viral, bacterial and eukaryotic model replisomes reconstituted in vitro
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