Biology subjects

Mohr, G.

Publications and source records attributed to Mohr, G..

3 recordsLinked to original sources

bioRxiv2025

A bacterial PrimPol-reverse transcriptase hybrid protein has a proofreading exonuclease activity that can be transferred to other reverse transcriptases

Gene disruption analysis revealed that an E. coli PPRT protein, which has an N-terminal Primase-Polymerase (PrimPol) domain fused to a group II intron-like reverse transcriptase (RT) domain followed by a long C-terminal domain (CTD), contributes to a cellular oxidative DNA damage response in addition to its previously described function in phage defense. Biochemical analysis showed that the PrimPol domain has an error-prone DNA polymerase activity that enables read through of oxidation-induced DNA damage. Surprisingly, we found that the RT-like domain, in addition to synthesizing protein-primed DNAs for phage defense, has a 3 to 5 DNA exonuclease activity that functions in proofreading DNAs synthesized by the PrimPol domain. Extending these findings, we identified structural features that contribute to this proofreading activity, enabling us to associate it with both a group II intron-encoded and retroviral RT and suggesting general methods for incorporating proofreading activity into RTs.

molecular biology↗

bioRxiv2023

Mechanisms used for cDNA synthesis and site-specific integration of RNA into DNA genomes by a reverse transcriptase-Cas1 fusion protein

Reverse transcriptase-Cas1 (RT-Cas1) fusion proteins found in some CRISPR systems enable spacer acquisition from both RNA and DNA, but the mechanism of RNA spacer acquisition has remained unclear. Here, we found Marinomonas mediterranea RT-Cas1/Cas2 adds short 3-DNA (dN) tails to RNA protospacers enabling their direct integration into CRISPR arrays as 3-dN-RNA/cDNA duplexes or 3-dN-RNAs at rates comparable to similarly configured DNAs. Reverse transcription of RNA protospacers occurs by multiple mechanisms, including recently described de novo initiation, protein priming with any dNTP, and use of short exogenous or synthesized DNA oligomer primers, enabling synthesis of cDNAs from diverse RNAs without fixed sequence requirements. The integration of 3-dN-RNAs or single-stranded (ss) DNAs is favored over duplexes at higher protospacer concentrations, potentially relevant to spacer acquisition from abundant pathogen RNAs or ssDNA fragments generated by phage-defense nucleases. Our findings reveal novel mechanisms for site-specifically integrating RNA into DNA genomes with potential biotechnological applications.

molecular biology↗

bioRxiv2022

Group II Intron-Like Reverse TranscriptasesFunction in Double-Strand Break Repair by Microhomology-Mediated End Joining

Bacteria encode free-standing reverse transcriptases (RTs) of unknown function that are closely related to group II intron-encoded RTs. Here, we found that a Pseudomonas aeruginosa group II intron-like RT (G2L4 RT) with YIDD instead of YADD at its active site functions in DNA repair in its native host and when transferred into Escherichia coli. G2L4 RT has biochemical activities strikingly similar to those of human DNA repair polymerase{theta} and uses them for translesion DNA synthesis and double-strand break repair (DSBR) via microhomology-mediated end-joining (MMEJ) in vitro and in vivo. We also found that a group II intron RT can function similarly to G2L4 RT in DNA repair, with reciprocal substitutions at the active site showing an I residue favors MMEJ and an A residue favors primer extension in both enzymes. The DNA repair functions of these enzymes utilize conserved structural features of non-LTR-retroelement RTs, including human LINE-1 and other eukaryotic non-LTR-retrotransposon RTs, suggesting such enzymes may have an inherent ability to function in DSBR in a wide range of organisms.

molecular biology↗