Construction of a highly error-prone DNA polymerase for developing organelle mutation systems

Abstract

A novel family of DNA polymerases replicates organelle genomes in a wide distribution of taxa encompassing plants and protozoans. Making error-prone mutator versions of gamma DNA polymerases revolutionised our understanding of animal mitochondrial genomes but similar advances have not been made for the organelle DNA polymerases present in plant mitochondria and chloroplasts. We tested the fidelities of error prone tobacco organelle DNA polymerases using a novel positive selection method involving replication of the phage lambda cI repressor gene. Unlike gamma DNA polymerases, ablation of 3′–5′ exonuclease function resulted in a modest 5–8-fold error rate increase. Combining exonuclease deficiency with a polymerisation domain substitution raised the organelle DNA polymerase error rate by 140-fold relative to the wild type enzyme. This high error rate compares favourably with error-rates of mutator versions of animal gamma DNA polymerases. The error prone organelle DNA polymerase introduced mutations at multiple locations ranging from two to seven sites in half of the mutant cI genes studied. Single base substitutions predominated including frequent A:A (template: dNMP) mispairings. High error rate and semi-dominance to the wild type enzyme in vitro make the error prone organelle DNA polymerase suitable for elevating mutation rates in chloroplasts and mitochondria.

Plant Organellar DNA Polymerases Evolved Multifunctionality through the Acquisition of Novel Amino Acid Insertions

“…POPs are classified in a novel class of DNAPs exclusively present in protozoan and plants. Protozoan, algae, and non-vascular plants harbor a single gene encoding for a POP, whereas POPs in flowering plants, with the exception of members of the Solanaceae family, are a duplicated gene [ 9 , 112 ]. The plant model A. thaliana contains two POPs: AtPOLIA and AtPOLIB.…”

Comparative transcriptome analysis identified ChlH and POLGAMMA2 in regulating yellow-leaf coloration in Forsythia

“…POLGAMMA2 encodes a nuclear-encoded organelle DNA polymerase responsible for the replication of mitochondria and plastid genome (Parent et al, 2011). POLGAMMA2, also known as POLYMERASE I A (Pol1A), is one of the two Plant Organellar DNA polymerases (POPs) in flowering plants (García-Medel et al, 2019;Ji and Day, 2020). POLGAMMA2 is mainly involved in DNA replication, while its paralog POLGAMMA1 serves an additional role in DNA repair (Parent et al, 2011;Morley et al, 2019).…”

“…Previous works showed that POLYMERASE I A (Pol1A) and POLYMERASE I B (Pol1B), designated as POLGAMMA2 and POLGAMMA1, respectively, were organellar DNA polymerases known as Plant Organellar DNA Polymerases (POPs). Most flowering plants harbor two POP genes, while the dicot Solanaceae species, 10.3389/fpls.2022.1009575 such as Solanum lycopersicum and Nicotiana tomentosiformis, contain only one POP gene (Ji and Day, 2020). It likely that the virus-induced silencing of FsPOLGAMMA2 severely disrupted the DNA replication within chloroplasts, leading to severely disrupted chloroplast development in tobacco and tomato.…”