Mitotic replisome disassembly depends on TRAIP ubiquitin ligase activity

Abstract

We have shown previously that the process of replication machinery (replisome) disassembly at the termination of DNA replication forks in the S-phase is driven through polyubiquitylation of one of the replicative helicase subunits (Mcm7) by Cul2LRR1 ubiquitin ligase. Interestingly, upon inhibition of this pathway in Caenorhabditis elegans embryos, the replisomes retained on chromatin were unloaded in the subsequent mitosis. Here, we show that this mitotic replisome disassembly pathway exists in Xenopus laevis egg extract and we determine the first elements of its regulation. The mitotic disassembly pathway depends on the formation of K6- and K63-linked ubiquitin chains on Mcm7 by TRAIP ubiquitin ligase and the activity of p97/VCP protein segregase. Unlike in lower eukaryotes, however, it does not require SUMO modifications. Importantly, we also show that this process can remove all replisomes from mitotic chromatin, including stalled ones, which indicates a wide application for this pathway over being just a “backup” for terminated replisomes. Finally, we characterise the composition of the replisome retained on chromatin until mitosis.

Mechanisms Generating Cancer Genome Complexity From A Single Cell Division Error

“…Access to replication factors, coupled with high mitotic cyclin-dependent kinase activity (51, 73), likely then activates replication on this incompletely replicated DNA. The DNA damage resulting from mitotic DNA replication may have a number of causes including the well-described activation of structure-specific endonucleases in mitosis (74) and/or the recently discovered cleavage of stalled DNA replication forks that occurs because of removal of the MCM2-7 replicative helicase from mitotic chromosomes (51, 75).…”

Under-Replicated DNA: The Byproduct of Large Genomes?

“…EdU incorporation in mitotic cells also depends on PLK1 (Polo Like Kinase 1), WAPL (Wings Apart like protein) and SMC2 (Structural maintenance of chromosomes protein 2), implying that MiDAS should occur after or in parallel with the release of sister chromatid arm cohesion [ 28 ]. Two recently published papers uncovered one more piece of the MiDAS’s puzzle: how cells switch from conventional S phase DNA replication to mitotic DNA replication [ 71 , 72 ]. The most likely MiDAS substrate is a stretch of UR-DNA which is formed after the processing of a DFS event.…”

“…These terminally arrested forks and their associated replisomes persist until mitosis and one possibility is that their presence might protect the parental DNA at stalled forks from premature nucleolytic attack. Mitotic replisome disassembly is driven by the polyubiquitination of the CMG component MCM7 by the E3-ubiquitin ligase TRAIP (TRAF-interacting protein) [ 71 , 72 , 73 ]. TRAIP-directed replisome disassembly is an early requirement for MiDAS.…”

“…Failure to perform MiDAS in the face of replicative stress channels the resolution of the UR-DNA to 53BP1-NBs in the following cell cycle. Indeed, the depletion or chemical inhibition of all the components required for—or related to—MiDAS, have shown to increase 53BP1-NBs in the following G1 phase: SLX4 [ 28 ], MUS81 [ 28 , 67 , 88 ], SMC2 [ 28 , 90 ], replicative polymerases [ 28 ], POLD3 [ 28 ], TOPBP1 [ 66 ], RAD52 [ 69 ], RECQ5 [ 68 ], TRAIP [ 71 , 72 ], BLM [ 90 ] and Polη [ 56 ]. Likewise, a considerable amount of UR-DNA due to high replicative stress experienced in the previous S phase could lead to the saturation of MiDAS and prompt its resolution in G1.…”

Working on Genomic Stability: From the S-Phase to Mitosis

“…Thus, TRAIP drives replisome disassembly at those under-replicated CFS loci, thereby allowing access to replication fork for the specific factors in charge for mitotic DNA synthesis [194]. In addition, the activity of TRAIP during mitosis seems to be regulated by Cdk1-Cyclin B1 complexes [56,194,195], which control the accurate timing of mitosis and also regulate the switch from conventional DNA replication to MiDAS machineries (Table 1). Together, these data suggest that the mechanisms of MiDAS have only just started to be unraveled.…”