Ubiquitylation at Stressed Replication Forks: Mechanisms and Functions

Abstract

Accurate duplication of chromosomal DNA is vital for faithful transmission of the genome during cell division. However, DNA replication integrity is frequently challenged by genotoxic insults that compromise the progression and stability of replication forks, posing a threat to genome stability. It is becoming clear that the organization of the replisome displays remarkable flexibility in responding to and overcoming a wide spectrum of fork-stalling insults, and that these transactions are dynamically orchestrated and regulated by protein post-translational modifications (PTMs) including ubiquitylation. In this review, we highlight and discuss important recent advances on how ubiquitin-mediated signaling at the replication fork plays a crucial multifaceted role in regulating replisome composition and remodeling its configuration upon replication stress, thereby ensuring high-fidelity duplication of the genome. Replication Stress and Ubiquitin SignalingPrecise and complete replication of cellular DNA during the S phase of each cell cycle is essential for genome stability, cell proliferation, and organismal fitness. The DNA replication process commences prior to S phase, when replication origins are licensed by the loading of inactive double minichromosome maintenance complex (MCM)2-7 hexamers [1-3]. In S phase, origin firing converts these double hexamers into active, bidirectional replication forks, through the recruitment of CDC45 and the GINS complex, leading to formation of the replicative CDC45-MCM2-7-GINS (CMG) helicase that translocates on the leading strand to unwind the duplex DNA template [4,5]. Although eukaryotic DNA replication initiates from multiple replication origins, only a fraction of licensed origins fire during a normal S phase. However, when obstacles that hinder replication fork progression are encountered, activation of otherwise dormant nearby origins provides an important rescue mechanism for completing genome duplication [6]. In S phase, the active replisome, consisting of the CMG helicase, replicative DNA polymerases, the replication factor C (RFC)-loaded clamp proliferating cell nuclear antigen (PCNA), and auxiliary factors, regulates every aspect of bidirectional replication, which occurs continuously on the leading strand and discontinuously on the lagging strand [7].Deregulation of DNA replication, giving rise to replication stress (see Glossary), is a hallmark of cancer cells and a recognized driver of genomic instability [8-10], representing an attractive target for clinical intervention. However, given the sheer size and complex organization of vertebrate genomes, low levels of replication stress occur naturally in most proliferating cells, arising due to obstacles including heterochromatin-imposed barriers (e.g., repetitive DNA sequences and G4 quadruplexes), replication-transcription collisions, ribonucleotide misincorporation, modified or mismatched nucleotides, and helix-distorting adducts that stall the advancing replication machinery [10]. Under normal conditions, such impe...

Lnc956-TRIM28-HSP90B1 complex on replication forks promotes CMG helicase retention to ensure stem cell genomic stability and embryogenesis

“…We also investigated the influence of HSP90B1 activity inhibition on MCM7 ubiquitylation. Immunoblotting analysis revealed that MCM7 proteins, which were enriched by immunoprecipitation through MCM7 antibody, underwent K48- and K63-ubiquitylation after HU treatment and the modification level increased with the stress intensity 41, 42 . Strikingly, inhibition of HSP90B1 activity with either inhibitor sharply increased both types of ubiquitylation levels (Fig.…”

The Hydrogen-Coupled Oligopeptide Membrane Cotransporter Pept2 is SUMOylated in Kidney Distal Convoluted Tubule Cells

“…A phosphorylation-dependent SUMO modification (PDSM) motif, composed of a SUMO consensus site and an adjacent proline-directed phosphorylation site (ΨKxExxSP), promotes SUMOylation is apparent in several regulators of gene expression, including heat shock factors (HSFs) and TFs ( Hietakangas et al, 2006 ). In contrast to ubiquitylation, a modification with many known functions ( Dittmar and Winklhofer, 2019 ; Deng et al, 2020 ; Zhu et al, 2020 ; Mirsanaye et al, 2021 ), the substantially greater number of SUMOylated TFs highlight the importance of SUMOylation for modulating DNA transcription. SUMOylation modulates the function of TFs through multiple mechanisms, including interaction with histone deacetylase complexes and other transcriptional coregulators, inhibition of acetylation and phosphorylation, as well as availability to bind chromatin ( Rosonina et al, 2017 ).…”

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“…Intriguingly, this complex molecule protects both fork integrity and promotes resistance of BRCA-deficient cells to PARP inhibitors. Based on this knowledge, it has been suggested that ubiquitinated PCNA may even operate parallel to the BRCA-RAD51 pathway to protect the replication fork progression [ 88 ]. Further experimental evidence made clear that, in response to DNA replication stress, the fork may reverse its direction thus providing time to start the repair procedure and preventing double-strand breaks [ 89 ].…”