Researcher(s)
- Ken Tran, Biochemistry, University of Delaware
Faculty Mentor(s)
- Zhihao Zhuang, Chemistry and Biochemistry, University of Delaware
Abstract
DNA damage tolerance (DDT) enables replication of DNA through lesions, such as abasic sites or UV-induced pyrimidine dimers, via two competing pathways: translesion synthesis (TLS) and template switching (TS).
TLS is triggered when the Rad6/Rad18 E2-E3 complex monoubiquitinates PCNA, a sliding clamp that confers high processivity to replicative polymerases in eukaryotic cells, at Lys164 (monoUb-PCNA). This recruits low-fidelity polymerases to replicate DNA past a lesion. TS offers an alternative route: the Rad5 E3 ligase, working alongside the Ubc13-Mms2 E2 heterodimer, extends monoUb-PCNA into K63-linked polyubiquitinated PCNA, thought to signal use of the undamaged sister strand as a template to bypass the lesion.
Template switching is thus an error-free DDT pathway that offers higher fidelity replication compared to error-prone TLS. However, the detailed mechanism of TS, and how cells alternate between these two pathways, remains poorly understood.
This work describes the semisynthesis of a fluorescently labeled monoubiquitinated PCNA probe designed to interact with ScRad5 (S. cerevisiae), enabling downstream binding and interaction assays that contribute to a better understanding of both TLS and template switching. Ubiquitin bearing an N-terminal pentaglycine tag is expressed, and its C-terminus is conjugated to a synthetic, non-hydrolyzable linker. This linker then joins with the K164C PCNA construct, in which all native cysteines are mutated to serine. A fluorophore containing an LPETG motif, recognized by Sortase A, is then ligated to the pentaglycine tag via sortase-mediated conjugation, yielding a labeled probe capable of reporting Rad5 engagement with monoUb-PCNA.
Loss of Rad5 activity in yeast increases cell reliance on the mutagenic TLS pathway. Its human ortholog, HLTF, has been implicated as a tumor suppressor, and reduced template-switching capacity has been linked to genomic instability. Understanding Rad5-PCNA engagement could therefore inform small-molecule designs that can disable DDT in cancer cells to combat their growth.