ABSTRACT Ultraviolet (UV) radiation generates crosslinked DNA lesions—primarily cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts (6–4 PPs)—that block the progression of replicative DNA polymerases. In plants, these lesions are efficiently removed from nuclear DNA by dedicated repair pathways; however, comparable repair mechanisms are absent in plastids and mitochondria. Consequently, how plant organellar DNA polymerases (POPs) tolerate or bypass UV-induced damage has remained unclear. Here, we show that the two Arabidopsis thaliana organellar polymerases, AtPolIs, possess robust translesion synthesis (TLS) activity across CPDs. Although wild-type enzymes display only limited extension across 6–4 PPs, removal of their exonuclease function dramatically enhances bypass, yielding an efficiency of replication across the 6–4 PP that closely resembles that observed on an undamaged template. This establishes AtPolI as the first known replicative DNA polymerase capable of efficiently bypassing a 6–4 PP. We further demonstrate that TLS across UV photoproducts relies on three unique amino acid insertions within the AtPolI polymerase domain, as deletion of any single insertion abolishes TLS. Notably, Mn²⁺ can restore TLS activity in these variants, but only for CPD lesions. Together, these findings identify AtPolIs as the first plant organellar replicases with intrinsic 6–4 PP bypass capability and define the structural features that enable this function. GRAPHICAL ABSTRACT
Baruch-Torres et al. (Tue,) studied this question.