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

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, Mn{superscript 2} 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 O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/701875v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@f497bcorg.highwire.dtl.DTLVardef@15ba2eorg.highwire.dtl.DTLVardef@724e29org.highwire.dtl.DTLVardef@697c8b_HPS_FORMAT_FIGEXP M_FIG C_FIG