The Ubiquitin Ligase TRAIP: Double-Edged Sword at the Replisome

Abstract

In preparation for cell division, the genome must be copied with high fidelity. However, replisomes often encounter obstacles, including bulky DNA lesions caused by reactive metabolites and chemotherapeutics, as well as stable nucleoprotein complexes. Here, we discuss recent advances in our understanding of TRAIP, a replisome-associated E3 ubiquitin ligase that is mutated in microcephalic primordial dwarfism. In interphase, TRAIP helps replisomes overcome DNA interstrand crosslinks and DNA-protein crosslinks, whereas in mitosis it triggers disassembly of all replisomes that remain on chromatin. We describe a model to explain how TRAIP performs these disparate functions and how they help maintain genome integrity.

Keywords: DNA interstrand crosslink repair; DNA-protein crosslink repair; E3 ubiquitin ligase; mitotic DNA replication; replication termination.

Figure 1.. Replication-coupled Interstrand crosslink (ICL) and DNA-protein crosslink (DPC) repair pathways.

A. In Xenopus egg extracts, three pathways of ICL repair require the convergence of two replication forks at the crosslink. Fork convergence triggers ubiquitylation of the replisome’s CDC45/MCM2-7/GINS (CMG) helicase by TRAIP (ubiquitin shown as purple spheres). Acetaldehyde-ICL repair is depicted as being initiated before CMG ubiquitylation (i), but this remains to be confirmed. Ubiquitin chains as short as one or two ubiquitins may recruit the NEIL3 glycosylase to directly unhook psoralen and abasic ICLs, allowing completion of DNA replication (ii). If NEIL3 fails to unhook the crosslink, TRAIP continues to extend the ubiquitin chains (iii). Long ubiquitin chains trigger CMG unloading by the p97 segregase, which allows Fanconi anemia pathway endonucleases to unhook the ICL. The resulting double-strand break is repaired by homologous recombination. B. Proteins crosslinked to the leading strand template hinder progression of the CMG helicase. Upon fork collision, TRAIP ubiquitylates the DPC (top arrow). After the accessory helicase RTEL1 promotes CMG bypass of the lesion, the DPC is ubiquitylated by a second, unknown E3 ubiquitin ligase. DPC ubiquitylation marks the lesion for proteolysis by the proteasome. The DPC-specific protease SPRTN also contributes to proteolysis of both ubiquitylated and unmodified DPCs. Note that at DPC-fork collisions, TRAIP activity does not require fork convergence and CMG is not ubiquitylated. In the absence of TRAIP, the initial ubiquitylation of the DPC is delayed, as is CMG bypass, but bypass does eventually occur, followed by CMG ubiquitylation by an unknown E3 ubiquitin ligase and proteolysis by SPRTN and the proteasome.

Figure 2.. Model for TRAIP function in S phase.

During unperturbed DNA replication in S phase, TRAIP is assembled with the replisome with its RING domain positioned to ubiquitylate any protein encountered by the replisome (inset; purple arrow signifies direction of ubiquitylation). Such proteins include DNA-protein crosslinks (DPCs) and CDC45/MCM2-7/GINS (CMG) helicases when forks converge at interstrand crosslinks (ICLs) (“S phase," center, gray). TRAIP ubiquitylation of the host replisome on which it is assembled (“S phase,” inset, gray broken arrow) does not occur, possibly because rigid positioning of the RING domain prevents it from bringing its E2 ubiquitin conjugating enzyme (brown) into contact with CMG. During mitosis, TRAIP undergoes a conformational change, allowing it to ubiquitylate its host CMG (“Mitosis,” purple arrow).

Figure 3.. Cell cycle regulation of TRAIP specificity.

A. (i) During S phase, the CDC45/MCM2-7/GINS (CMG) helicases of stalled but unconverged replisomes are not ubiquitylated. However, if these stalled CMGs persist into mitosis, they undergo TRAIP-dependent ubiquitylation and subsequent unloading by p97. (ii) Terminated CMGs are normally ubiquitylated in S phase by the Cul2-based RING E3 ubiquitin ligase CRL2^Lrr1. (iii) in the absence of CRL2^Lrr1, terminated CMGs remain unmodified and persist on chromatin until mitosis, when they are ubiquitylated by TRAIP and unloaded. B. (i) During mitosis, CMGs stalled at the boundaries of a common fragile site are ubiquitylated by TRAIP. (ii) Ubiquitylated CMGs are unloaded, which exposes the leading strand templates of the two forks to structure-specific endonucleases such as MUS81-EME1. (iii) Cleavage of the leading strand templates results in two broken ends (highlighted in red) and an intact strand (highlighted in blue). The two broken ends are repaired by end joining (iv), while the intact strand is restored by gap filling (green arrows) (v). These two repair processes result in the deletion of the unreplicated DNA and sister chromatid exchange, which are hallmarks of common fragile site (CFS) expression. C. If a chromosome containing a stalled fork enters mitosis in the absence of a nearby converging fork (e.g. at a telomere) (i), TRAIP-mediated mitotic CMG unloading (ii) leads to leading strand template cleavage and a single-ended double-strand break (highlighted in red) (iii). This break could then be repaired by break-induced replication (iv).