DNA polymerase I and DnaE mediate RNA-templated DNA synthesis over embedded ribonucleotides

It is established that during normal growth and under stress, ribonucleotides become nested in genomic DNA. Given the high frequency with which RNA is found in DNA, it is unknown which bacterial DNA polymerases are responsible for catalyzing RNA-dependent DNA synthesis. In this work, we establish bacterial DNA polymerase I (Pol I), a robust reverse transcriptase (RT), as the primary bypass enzyme. We tested several bacterial Pol I enzymes and found that all possess RT activity, with Bacillus subtilis Pol I showing robust RNA-dependent DNA synthesis activity similar to the Moloney murine leukemia virus RT. In contrast, template RNA slowed or blocked DNA synthesis by B. subtilis replicative C-family DNA polymerases PolC and DnaE. We tested several substrates with increasing patches of consecutive ribonucleotides in DNA and found that a single ribonucleotide slows synthesis by PolC, while DnaE can traverse a patch of five consecutive ribonucleotides. Therefore, DnaE activity would allow for ribonucleotide bypass of short patches, while Pol I could bypass longer stretches up to 40 nt in length. We conclude that Pol I and DnaE are responsible for RNA-templated DNA replication, establishing the DNA polymerase hierarchy for navigating RNA tracts that become integrated into genomic DNA.