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Discrimination against the Fluorescent Cytosine Analog tC by Escherichia coli DNA Polymerase IV DinB

Y family DNA polymerases are known for their ability to accommodate bulky lesions and to replicate beyond such lesions via translesion synthesis. Fluorescent nucleotide analogs are used to study the structure and dynamics of nucleic acids, the dynamics of DNA polymerase activity, and damage tolerance and repair. An analog of cytosine, 1,3‐diaza‐2‐oxophenothiazine (tC), has been used to study the polymerase domain of the A family polymerase known as the Klenow fragment. The Klenow fragment can efficiently incorporate the deoxytriphosphate‐tC into the growing strand, and bypass it as it encounters it on the template strand. We probed the ability of the Y family DNA polymerase DinB to copy DNA containing tC and to incorporate tC into a growing DNA strand. We demonstrate that DinB can add dGTP across from tC in template DNA, but DinB cannot extend beyond the newly‐formed base pair. DinB can also incorporate the tC triphosphate across from G in the template strand, but can only add one nucleotide beyond that. This is intriguing as Y family polymerases have open active sites and are thought to be more accommodating than replicative polymerases. We find that the Y family polymerase DinB discriminates against tC in the template strand, which may suggest that DinB specifically discriminates against modifications in the major groove of DNA. Support: NSF Career MCB‐0845033 & RCSA