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.

Keywords: DPC; DPC proteolysis; Ruijs-Aalfs syndrome; SPRTN protease; translesion synthesis (TLS).

FIGURE 1

Types of DPCs. Schematic depicting the four types of DPCs. (A) Type 1 DPCs are proteins crosslinked to unperturbed duplex DNA, generally induced by nonspecific agents. (B) Type 2 DPCs are crosslinked to the ends of SSBs and arise from abortive DNA repair processes. (C) Types 3 and 4 DPCs are abortive DNA-topoisomerase DPCs, where TOP1 is crosslinked to the 3ʹ end of a SSB or TOP2 is crosslinked to the 5ʹ ends of a DSB via a phosphotyrosyl bond. Adapted from Nakano et al. (2017), Stingele et al. (2017). Created in BioRender.com.

FIGURE 2

Mechanisms of DPC degradation. Schematic depicting pathways for debulking of DPCs in (A) replication-independent and (B) replication-dependent contexts. (A) Outside of DNA replication, DPCs are targeted for SUMOylation by an E3 SUMO ligase such as PIAS4. Subsequent DPC recognition and polyubiquitination by a SUMO-targeted E3 ubiquitin ligase such as RNF4 promotes proteasomal degradation of the DPC to allow for downstream repair of DNA breaks (Sun et al., 2020c; Liu et al., 2021). (B) During DNA replication, fork collision with a DPC triggers helicase-polymerase uncoupling by CMG helicase bypass of the lesion. The DPC may be targeted for modification by ubiquitin or SUMO. Polyubiquitinated DPCs can be targeted for proteasomal degradation. Alternatively, if the polymerase extends the nascent DNA to within a few nucleotides of the lesion, SPRTN-mediated DPC proteolysis is activated to degrade modified or unmodified DPCs. The remaining peptide-DNA adduct can be bypassed by TLS polymerases and repaired post-replication (Larsen et al., 2019; Sparks et al., 2019; Ruggiano et al., 2021). Created in BioRender.com.

FIGURE 3

SPRTN is a multidomain protein. Schematic depicting the domains of SPRTN and Wss1 metalloproteases. Small numbers represent aa ranges of indicated domains. (A) SprT = metalloprotease domain; SH = VCP-interacting motif; PIP = PCNA-interacting protein box motif; UBZ = ubiquitin binding domain. The SprT domain can be subdivided into the canonical metalloprotease domain (aa 45–166), which contains the conserved HEXXH active site, and the Zn²⁺-binding subdomain which assists in substrate cleavage and has secondary DNA binding function (aa 167–212) (Li et al., 2019). The basic region (aa 220–230) is a critical DNA binding region (Toth et al., 2017; Li et al., 2019). Identified SPRTN auto-cleavage sites are indicated with green lollipops (Vaz et al., 2016), and identified phosphosites are indicated with magenta lollipops (Halder et al., 2019). (B) WLM = metalloprotease domain; SH/VIM = VCP-interacting motif; SIM = SUMO-interaction motif (Vaz et al., 2017). Created in BioRender.com.

FIGURE 4

Mutations in SPRTN cause Ruijs-Aalfs syndrome. Schematic depicting SPRTN protein products resulting from biallelic mutations identified in RJALS patients. Family A mutants resulted in a premature STOP codon and SPRTN truncation at aa 249 (SPRTN ΔC). Family B compound heterozygous mutations resulted in aa substitution at tyrosine 117 mutated to cysteine (SPRTN Y117C), and a premature STOP codon and truncation at aa 246. RJALS causes early-onset hepatocellular carcinoma and premature aging (Lessel et al., 2014). Created in BioRender.com.