Debulking of topoisomerase DNA-protein crosslinks (TOP-DPC) by the proteasome, non-proteasomal and non-proteolytic pathways

Abstract

Topoisomerases play a pivotal role in ensuring DNA metabolisms during replication, transcription and chromosomal segregation. To manage DNA topology, topoisomerases generate break(s) in the DNA backbone by forming transient enzyme-DNA cleavage complexes (TOPcc) with phosphotyrosyl linkages between DNA ends and topoisomerase catalytic tyrosyl residues. Topoisomerases have been identified as the cellular targets of a variety of anti-cancer drugs (e.g. topotecan, irinotecan, etoposide and doxorubicin, and antibiotics (e.g. ciprofloxacin and levofloxacin). These drugs, as well as other exogenous and endogenous agents, convert the transient TOPcc into persistent TOPcc, which we refer to as topoisomerase DNA-protein crosslinks (TOP-DPC) that challenge genome integrity and lead to cell death if left unrepaired. Proteolysis of the bulky protein component of TOP-DPC (debulking) is a poorly understood repair process employed across eukaryotes. TOP-DPC proteolysis can be achieved either by the ubiquitin-proteasome pathway (UPP) or by non-proteasomal proteases, which are typified by the metalloprotease SPRTN/WSS1. Debulking of TOP-DPC exposes the phosphotyrosyl bonds, hence enables tyrosyl-DNA phosphodiesterases (TDP1 and TDP2) to access and cleave the bonds. In this review, we focus on current knowledge of the protease pathways for debulking TOP-DPC and highlighting recent advances in understanding the mechanisms regulating the proteolytic repair pathways. We also discuss the avenues that are being exploited to target the proteolytic repair pathways for improving the clinical outcome of topoisomerase inhibitors.

Keywords: DNA-protein crosslinks; SPRTN; SUMOylation; Topoisomerases; Ubiquitin-proteasome pathway.

Fig. 1.. Pathways for the debulking of TOP-DPC.

Overall scheme for the induction and excision repair of TOP-DPC. The ubiquitin proteasomal pathway (UPP) is represented in the middle with the different ubiquitin pathways for TOP-DPC (Cullins, SUMO-targeted ubiquitin ligases [STUbLs]). The non-proteasomal pathway is examplifed by Spartan/Wss1. Both the proteasomal and non proteasomal generate DNA peptide crosslinks (DpC). The non-proteolytic pathways shown as the arrow at right includes the endonuclease pathway (examplified by MRE11) and the ZATT^ZNF451 pathway, which unfolds TOP2-DPC and primes their hydrolytic removal by TDP2 [125]. TOP-DPC excision is detailed in a separate issue of DNA Repair [1].

Fig. 2.. Architecture and polarity of TOP-DPC in the context of the enzymatic DPC.

Pathological enzymatic DPC result from abortive enzymatic reactions. Depending on the structure of the DNA at the DPC site, enzymatic DPC can be classified in two groups: (a) DPC with intact DNA backbone as in the case of DNMT (DNA methyltransferase) covalently linked to 5-azacytosine incorporated into the DNA or HMCES (5-hydroxymethylcytosine binding ES specific) at AP sites in single-stranded DNA, and (b) DPC at the end of a nick, or terminal DPC, which can be subclassified as 5’-DPC and 3’-DPC depending whether the DPC is at the 5’- or 3’-terminus of the DNA (see text for details). DNA polymerase β (POLB) forms 5’-DPC whereas tyrosyl-DNA-phosphodiesterase 1 (TDP1) and poly(ADPribose)polymerase 1 (PARP1) form 3’-DPC. TOP-DPC belong to both the 5’- and 3’-DPC subgroups as they generate transient covalent protein-linked DNA breaks (PLDB), termed topoisomerase cleavage complexes (TOPcc) at either the 5’- or 3’-end depending on the topoisomerase. Abortive (trapped) TOPcc form stable TOP-DPC at the end of the broken DNA. c. TOP1 and TOP1MT cleaves one strand of duplex DNA with covalent linkage through their active tyrosine (Y) to the 3’-end. d. TOP2 enzymes (TOP2α [TOP2A] and TOP2β [TOP2B]) and the meiotic recombination topoisomerase-like SPO11 act as homodimers and cleave duplex DNA with covalent linkage through their active tyrosine (Y). e. TOP3 enzymes (TOP3α (TOP3A) and TOP3β [TOP3B]) act as monomers. They reversibly cleave exposed single-stranded DNA regions of the DNA double helix by covalent linkage to the 5’-end using Mg²⁺. Little is known about the repair of TOP3-DPC (see Fig. 4 and text for details).

Fig. 3.. The ubiquitin-proteasome system (UPP).

a. Ubiquitin (Ub) is a small protein (~76 amino acid) forming a covalent isopeptide bond between its c-terminal glycine carboxyl group and the NH2 group of a lysine residue on either substrate proteins or ubiquitin itself. b. Substrate ubiquitylation is achieved in three steps: substrate proteins are processed by ATP-activated enzymatic cascades with ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin ligases (E3). Ub is first activated by an E1 in an ATP-dependent manner. The E1 catalyzes the acyl-adenylation of the C-terminus of an ubiquitin molecule using ATP. The activated Ub is then conjugated to E1 through a thioester bond between the c-terminal carboxyl group of ubiquitin and the E1 cysteine sulfhydryl group (activation). E2 enzymes transfers the Ub molecule from E1 to the active site cysteine of an E2 via a trans(thio)esterification reaction (conjugation). E3 ligases allow the attachment of the Ub molecule from E2 to a substrate protein (S) by catalyzing the formation of an isopeptidyl linkage between a lysine residue of the substrate and the c-terminal glycine of the ubiquitin (ligation). Lysine 48-linked polyubiquitylation targets substrates to the 26S proteasome. The 19S regulatory particle of the proteasome docks the ubiquitylated substrate, unfolds its and deubiquitylates the substrate to recycle ubiquitin and transfer it to the 20S core particle where 3 proteolytic subunits degrade the substrate (see Fig. 4).

Fig. 4.. Known UPP repair pathways for TOP-DPC.

a-b. DNA translocating complexes including transcription (a) and replication (b) trigger the ubiquitylation and subsequent proteasomal degradation of TOP-DPC. Cullin-RING ligases have been implicated as the E3 for both TOP1- and TOP2-DPC. c. An alternative ubiquitylation pathway is by STUbL (SUMO-targeted Ub ligases), which are recruited by SUMOylation of the TOP1- and TOP2-DPC. SUMOylation-dependent ubiquitylation is catalyzed by SUMO ligase PIAS4 and STUbL RNF4 in human cells and Siz1 and Slx5/Slx8 in yeast. d. Recent studies show that TOP3β-DPC are directly ubiquitylated by the E3 ligase TRIM41 (see text for details and reference).

Fig. 5.. Repair of TOP-DPC by the non-proteasomal metalloproteases SPRTN/Wss1.

a. Domain/motif architecture of human SPRTN and its yeast ortholog Wss1. ZBD, Zinc binding domain; PIP, PCNA interaction peptide; SHP, p97 or VCP-binding motif; SIM, SUMO interaction motif; SprT, the metalloprotease domain similar to that of the Escherichia coli SprT protein; UBZ, ubiquitin-binding zinc finger; VIM; VCP interaction motif; WLM, Wss1- like metalloprotease domain Schematic of the repair of TOP-DPC by SPRTN/Wss1. b. As a component of the replisome, SPRTN/WSS1 is activated by its binding to ssDNA. It targets TOP-DPC as well as general DPC for proteolysis in association with replication.