A Bacteriophage‐Derived Primase‐Helicase Orchestrates Plant Organellar <scp>DNA</scp> Replication

The mechanisms underlying the assembly and regulation of enzymatic complexes responsible for plant organellar DNA replication remain poorly characterized. Unlike the monophyletic origin of the gene products involved in animal mitochondrial replication, derived from T-odd bacteriophages, plant organellar DNA replication relies on genes either unique to plants or with origins traceable to bacteria and bacteriophages. Here, we demonstrate that the bacteriophage-related primase-helicase from Arabidopsis thaliana (AtTwinkle) is essential for double-stranded DNA unwinding. AtTwinkle functionally interacts with bacterial-related organellar DNA polymerases (AtPolIs), which lack the ability to unwind large regions of dsDNA, coupling DNA unwinding to processive DNA synthesis at the leading strand of the replisome. Analysis of two T-DNA insertion mutants of AtTwinkle reveals distinct phenotypic outcomes; these mutant lines are hereafter referred to as ph. The ph1 (-/-) mutant, which carries a T-DNA insertion in the 5´ UTR region, is viable and exhibits no noticeable developmental differences compared to wild-type plants. In contrast, the ph2 mutant, with a T-DNA insertion in the 19th exon, displays embryo lethality. Despite these differences, both ph1 (-/-) and heterozygous ph2 (+/-) mutants show a reduction in organellar DNA copy numbers under non-stress conditions and exhibit heightened sensitivity to DNA-damaging agents. In summary, our findings demonstrate that AtTwinkle is essential for organellar DNA replication. The heightened sensitivity of insertion mutants to organelle-specific genotoxic agents indicates that loss of AtTwinkle function reduces the availability of template DNA necessary for double-strand break (DSB) repair. Collectively, our findings reveal that two proteins of distinct evolutionary origins-AtTwinkle and plant organellar DNA polymerases-coevolved to coordinate DNA replication in plant organelles.