Sequence context controls the DNA binding and processivity of the T7 DNA primase

Primases are key enzymes involved in DNA replication. They act on single-stranded DNA, and catalyze the synthesis of short RNA primers used by DNA polymerases. Here, we investigate the DNA-binding and functional activity of the bacteriophage T7 primase using a new workflow called High-Throughput Primase Profiling (HTPP). Using a unique combination of high-throughput binding assays and biochemical analyses, HTPP reveals a complex landscape of binding specificity and functional activity for the T7 primase, determined by sequences flanking the primase recognition site. We identified specific features, such at G/T-rich flanks, which in-crease primase-DNA binding up to 10-fold and, surprisingly, also increase the length of newly formed RNA (2-3 fold). To our knowledge, variability in primer length has not been reported for this primase. We expect that applying HTPP to additional enzymes will reveal new insights into the effects of DNA sequence composition on the DNA recognition and functional activity of primases. HTPP (High-Throughput Primase Profiling) experiments were performed for recombinant T7 primase residue: 1-271. Briefly, the HTPP involved binding of his-tagged primase to single-stranded 180K Agilent microarrays in order to determine their binding specificity in different artificial context. Each sequence represented on the array is present in 6 replicate spots. We report the signal intensity for each spot.