AUTHOR=Huan Yang Wei , Brown Russell , Wang Baojun 

TITLE=An adenine/thymidine-rich region is integral to RepL-mediated DNA replication

JOURNAL=Frontiers in Microbiology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2023.1095671

DOI=10.3389/fmicb.2023.1095671

ISSN=1664-302X

ABSTRACT=<p>The lytic replication of bacteriophage P1 requires RepL expression and the lytic stage origin, oriL, which is postulated to be located within <italic>repL</italic> gene sequence. The exact sequence of P1 oriL and the mechanism(s) of RepL-mediated DNA replication, however, are not fully understood. By using <italic>repL</italic> gene expression to induce DNA replication of a <italic>gfp</italic> and a <italic>rfp</italic> reporter plasmids, we demonstrated that synonymous base substitution in an adenine/thymidine-rich region of <italic>repL</italic> gene sequence, termed AT2, significantly inhibited the RepL-mediated signal amplification. Contrastingly, mutations in an IHF and two DnaA binding sites did not affect the RepL-mediated signal amplification significantly. A truncated <italic>repL</italic> sequence with the AT2 region allowed RepL-mediated signal amplification <italic>in trans</italic> therefore verifying a significant role of the AT2 region in RepL-mediated DNA replication. A combination of <italic>repL</italic> gene expression and a non-protein-coding copy of <italic>repL</italic> gene sequence (termed nc-<italic>repL</italic>) was able to amplify the output of an arsenic biosensor. Furthermore, mutation(s) at single or multiple positions within the AT2 region produced varying levels of RepL-mediated signal amplification. Overall, our results provide novel insights into the identity and location of P1 oriL as well as demonstrating the potential of using <italic>repL</italic> constructs to amplify and modulate the output of genetic biosensors.</p>