Mechanisms and Regulation of DNA-Protein Crosslink Repair During DNA Replication by SPRTN Protease

Abstract

DNA-protein crosslinks (DPCs) are deleterious DNA lesions that occur when proteins are covalently crosslinked to the DNA by the action of variety of agents like reactive oxygen species, aldehydes and metabolites, radiation, and chemotherapeutic drugs. Unrepaired DPCs are blockades to all DNA metabolic processes. Specifically, during DNA replication, replication forks stall at DPCs and are vulnerable to fork collapse, causing DNA breakage leading to genome instability and cancer. Replication-coupled DPC repair involves DPC degradation by proteases such as SPRTN or the proteasome and the subsequent removal of DNA-peptide adducts by nucleases and canonical DNA repair pathways. SPRTN is a DNA-dependent metalloprotease that cleaves DPC substrates in a sequence-independent manner and is also required for translesion DNA synthesis following DPC degradation. Biallelic mutations in SPRTN cause Ruijs-Aalfs (RJALS) syndrome, characterized by hepatocellular carcinoma and segmental progeria, indicating the critical role for SPRTN and DPC repair pathway in genome maintenance. In this review, we will discuss the mechanism of replication-coupled DPC repair, regulation of SPRTN function and its implications in human disease and cancer.

O6-Alkylguanine-DNA Alkyltransferase Maintains Genomic Integrity During Peroxynitrite-Mediated DNA Damage by Forming DNA-Protein Crosslinks

“…To determine the mechanism behind this novel protective function of hAGT, pEGFP-N1 plasmid DNA having the kanamycin-resistant gene was exposed to PN (250 µM) on ice for 10 min followed by incubation with buffer, hAGT or C145S-hAGT protein for 2 h at 37 °C. [45] Because of the recent discovery of DNA-stimulated nuclear proteases that digest DPCs into DNA-peptide crosslinks and our speculation of a possible DNA-hAGT crosslink formation,[34, 46] we digested the reaction mixture with trypsin before transforming it into E. coli . Transformed E. coli cells were plated on kanamycin-containing LB agar plates and incubated overnight at 37 °C.…”

“…We also performed parallel experiments with MDA-MB-231 mammalian cell line with the exception of trypsin digestion of the reaction mixture before transfection due to the presence of DNA-stimulated proteases in mammalian cells. [34] Here, the functional integrity of the pEGFP-N1 plasmid DNA was measured by its ability to produce green fluorescence through the expression of EGFP which renders the cells green when visualized with the FITC filter of an epifluorescence microscope. The results were similar to that obtained with the E. coli experiment (Figure 2C).…”

“…This is because DPCs are bulky lesions that present a challenge for the cellular repair systems and often thought to cause replication block. However, the presence of DNA-stimulated proteases that can digest bulky DPCs[33, 34], translesion polymerases that can bypass DNA-peptide crosslinks,[33] and ability of the cell to repair DNA-peptide crosslinks, probably through NER and HR,[57] suggest otherwise. In fact, the hAGT-DNA crosslink provides a way by which the PN-generated 8-NO 2 -G lesions are shielded from being spontaneously converted into abasic sites and strands breaks by the cell, till repair sets in.…”

Recurrent duplication and diversification of a vital DNA repair gene family across Drosophila

“…To restore healthy rates of DNA-protein crosslink repair, regulation of the local Spartan protein dose may evolve though multiple mechanisms. Changes to transcript and/or protein abundance, protein affinity for the replication fork, and/or self-cleavage of excess Spartan protein (Perry and Ghosal 2022) can each alter Spartan protein dose and rebalance DNA-protein crosslink repair rates.…”

Editorial: The repair of DNA–protein crosslinks

“…SPRTN is a replication-coupled protease that digests both break-associated DPC substrates (e.g., topoisomerase DPCs or TOP-DPCs) and DPC substrates without DNA breaks in a sequence-independent manner. Biallelic mutations in SPRTN have been shown to be implicated in Ruijs-Aalfs (RJALS) syndrome, characterized by hepatocellular carcinoma and segmental progeria (Perry and Ghosal, 2022). The 26S proteasome targets DPCs through polyubiquitylation of the substrates throughout the cell cycle.…”