Translesion DNA synthesis on pyrimidine dimers by plant organellar DNA polymerases is metal-dependent.

Ultraviolet (UV) radiation generates DNA lesions, primarily cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts ([6-4] PPs), that can block DNA replication. Although nuclear UV-induced lesions are repaired or bypassed by specialized pathways, how plant organellar DNA polymerases replicate UV-damaged templates remains unclear. Here, we show that the two Arabidopsis thaliana organellar DNA replicases, AtPolIs, efficiently synthesize across CPDs with 80%-90% bypass efficiency, exceeding that reported for individual specialized translesion synthesis (TLS) polymerases. Furthermore, although [6-4] PPs impose a major barrier to most TLS polymerases, wild-type AtPolIs exhibit measurable lesion-bypass activity (∼10%), and reduction of exonuclease activity enhances bypass by ~8-fold, reaching levels comparable to synthesis on undamaged templates. We further demonstrate that TLS across UV photoproducts depends on three unique amino acid insertions within the polymerase domain, as disruption of these insertions severely compromises lesion bypass. These findings reveal that AtPolIs are replicative polymerases with an intrinsic and unusually robust capacity for UV-lesion bypass, suggesting a specialized adaptation that helps maintain plant organellar genome stability under UV stress.