Plant organellar DNA polymerases bypass thymine glycol using two conserved lysine residues

Abstract

Plant organelles cope with endogenous DNA damaging agents, byproducts of respiration and photosynthesis, and exogenous agents like ultraviolet light. Plant organellar DNA polymerases (DNAPs) are not phylogenetically related to yeast and metazoan DNAPs and they harbor three insertions not present in any other DNAPs. Plant organellar DNAPs from Arabidopsis thaliana (AtPolIA and AtPolIB) are translesion synthesis (TLS) DNAPs able to bypass abasic sites, a lesion that poses a strong block to replicative polymerases. Besides abasic sites, reactive oxidative species and ionizing radiation react with thymine resulting in thymine glycol (Tg), a DNA adduct that is also a strong block to replication. Here, we report that AtPolIA and AtPolIB bypass Tg by inserting an adenine opposite the lesion and efficiently extend from a Tg-A base pair. The TLS ability of AtPolIB is mapped to two conserved lysine residues: K593 and K866. Residue K593 is situated in insertion 1 and K866 is in insertion 3. With basis on the location of both insertions on a structural model of AtPolIIB, we hypothesize that the two positively charged residues interact to form a clamp around the primer-template. In contrast with nuclear and bacterial replication, where lesion bypass involves an interplay between TLS and replicative DNA polymerases, we postulate that plant organellar DNAPs evolved to exert replicative and TLS activities.

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

“…Since AtPOLIs are the sole DNAPs in mitochondria and chloroplasts, it is possible that the ability to bypass DNA lesions is directly linked to the acquisition of those insertions. Besides TLS at AP sites, AtPOLIs execute TLS across thymine glycol, preferentially by incorporating dAMP opposite to the lesion [20]. As is the case for an AP site, insertion 1 and insertion 3 of AtPOLIs are responsible for TLS [20].…”

“…Besides TLS at AP sites, AtPOLIs execute TLS across thymine glycol, preferentially by incorporating dAMP opposite to the lesion [20]. As is the case for an AP site, insertion 1 and insertion 3 of AtPOLIs are responsible for TLS [20].…”

“…HsDNAP executes TLS because of a single arginine residue (R2254) located in insertion 2 [21]. Sequence analysis of several POPs shows that insertion 1 is particularly rich in lysines [17,20]. AtPOLIB holds seven lysines in this insertion (K590, K593, K599, K605, K606, K610, and K615), whereas insertion 3 harbors two lysines (K859 and K866) [20].…”

“…Because of their singularity, these DNAPs POPs are the sole DNAPs localized in plant organelles and in A. thaliana, these polymerases have evolved to function in both DNA replication and translesion DNA synthesis (TLS) [17,19]. Both AtPOLIs efficiently bypass AP sites and thymine glycol, even in the presence of an active 3 -5 exonuclease domain [17,20]. The most notorious characteristic of AtPOLIs, in comparison to bacterial DNAPs, is the presence of three insertions in their polymerization domain.…”

“…The most notorious characteristic of AtPOLIs, in comparison to bacterial DNAPs, is the presence of three insertions in their polymerization domain. Insertions 1 and 2 are located in the thumb subdomain and a homology model suggests that they are poised to interact with double-stranded DNA, whereas insertion 3 is localized at the finger´s subdomain (Figure 1) [17,20]. The abilities to execute lesion bypass in AtPOLIs reside in insertions 1 and 3.…”