TOPBP1 recruits TOP2A to ultra-fine anaphase bridges to aid in their resolution.

During mitosis, sister chromatids must be faithfully segregated to ensure that daughter cells receive one copy of each chromosome. However, following replication they often remain entangled. Topoisomerase IIα (TOP2A) has been proposed to resolve such entanglements, but the mechanisms governing TOP2A recruitment to these structures remain poorly understood. Here, we identify TOPBP1 as a novel interactor of TOP2A, and reveal that it is required for TOP2A recruitment to ultra-fine anaphase bridges (UFBs) in mitosis. The C-terminal region of TOPBP1 interacts with TOP2A, and TOPBP1 recruitment to UFBs requires its BRCT domain 5. Depletion of TOPBP1 leads to accumulation of UFBs, the majority of which arise from centromeric loci. Accordingly, expression of a TOPBP1 mutant that is defective in TOP2A binding phenocopies TOP2A depletion. These findings provide new mechanistic insights into how TOP2A promotes resolution of UFBs during mitosis, and highlights a pivotal role for TOPBP1 in this process.We thank Drs G. Stewart and F. Esashi for cell lines, Professor T.D. Halazonetis, Dr G.J. Gorbsky and Dr G. Stewart for plasmids and antibodies. We also thank Dr C. Lagerholm (Wolfson Imaging Centre, Oxford) and Dr D. Waithe (CBRG, Oxford) for their help with microscopy and image analysis, and the Mass Spectrometry Laboratory (IBB PAS) for their work on analyses of GFP–TOP2A immunoprecipitation experiments. We also thank Professor I. Hickson for helpful comments on the manuscript. This work was funded by a Worldwide Cancer Research International Fellowship (to W.N.), a WIMM/Medical Research Council Senior Non-Clinical Fellowship (to W.N.), a Polish Ministry of Science and Higher Education fellowship (to J.N.) and Polish National Science Center grant N N303 571539 (to J.N.).This is the final published version. It first appeared at http://www.nature.com/ncomms/2015/150312/ncomms7572/full/ncomms7572.html#abstract

Ctf18-RFC and DNA Pol ϵ form a stable leading strand polymerase/clamp loader complex required for normal and perturbed DNA replication

The eukaryotic replisome must faithfully replicate DNA and cope with replication fork blocks and stalling, while simultaneously promoting sister chromatid cohesion. Ctf18-RFC is an alternative PCNA loader that links all these processes together by an unknown mechanism. Here, we use integrative structural biology combined with yeast genetics and biochemistry to highlight the specific functions that Ctf18-RFC plays within the leading strand machinery via an interaction with the catalytic domain of DNA Pol ϵ. We show that a large and unusually flexible interface enables this interaction to occur constitutively throughout the cell cycle and regardless of whether forks are replicating or stalled. We reveal that, by being anchored to the leading strand polymerase, Ctf18-RFC can rapidly signal fork stalling to activate the S phase checkpoint. Moreover, we demonstrate that, independently of checkpoint signaling or chromosome cohesion, Ctf18-RFC functions in parallel to Chl1 and Mrc1 to protect replication forks and cell viability. [Abstract copyright: © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

A Decade of Discovery—Eukaryotic Replisome Disassembly at Replication Termination

Simple Summary: During cell division, DNA is duplicated through a process called DNA replication, so that each new cell inherits a copy of its own. A high level of accuracy is essential in this for the maintenance of genome stability and the prevention of genetic disorders and ageing-related diseases. In this review, we describe the current knowledge around DNA replication termination, in particular comparing and contrasting the proteins and mechanisms identified in different organisms—from archaea through to humans—but with a specific focus upon eukaryotic replication termination. We discuss when and where termination takes place, the mechanisms of replication fork convergence and the process of replisome disassembly, in both S-phase and in mitosis. Recent advances in the field have revealed high levels of regulation in the process of replisome disassembly, demonstrating the importance of timely and appropriate unloading of replication machinery. Finally, we summarise how replication termination defects may impact cellular health and raise questions to be addressed in the future within the field. Abstract: The eukaryotic replicative helicase (CMG complex) is assembled during DNA replication initiation in a highly regulated manner, which is described in depth by other manuscripts in this Issue. During DNA replication, the replicative helicase moves through the chromatin, unwinding DNA and facilitating nascent DNA synthesis by polymerases. Once the duplication of a replicon is complete, the CMG helicase and the remaining components of the replisome need to be removed from the chromatin. Research carried out over the last ten years has produced a breakthrough in our understanding, revealing that replication termination, and more specifically replisome disassembly, is indeed a highly regulated process. This review brings together our current understanding of these processes and highlights elements of the mechanism that are conserved or have undergone divergence throughout evolution. Finally, we discuss events beyond the classic termination of DNA replication in S-phase and go over the known mechanisms of replicative helicase removal from chromatin in these particular situations

A mild and efficient approach to the 6H-oxazolo[3,2-f]pyrimidine-5,7-dione scaffold via unexpected rearrangement of 2,3-dihydropyrimido[6,1-b][1,5,3]dioxazepine-7,9(5H,8H)-diones:synthesis, crystallographic studies, and cytotoxic activity screening

We report a mild and efficient approach to the optically pure 6H-oxazolo[3,2-f]pyrimidine-5,7-dione scaffold via the unexpected rearrangement and ring contraction of 2,3-dihydropyrimido[6,1-b][1,5,3]- dioxazepine-7,9(5H,8H)-diones derived from nucleoside precursors. The developed procedure enables the synthesis of a wide range of compounds with great structural diversity. The structure of the obtained compounds was confirmed by NMR spectroscopy and single crystal X-ray structural analysis. The final products were tested for cytotoxic effect on one non-cancerous (fibroblasts) and six cancer cell lines of different origins (colon, glioma, breast, cervix, vulvar, and lung). The synthesized products are low molecular weight compounds with lead-like properties suitable for a medicinal chemistry optimization program

The S phase checkpoint promotes the Smc5/6 complex dependent SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε

Replication fork stalling and accumulation of single-stranded DNA trigger the S phase checkpoint, a signalling cascade that, in budding yeast, leads to the activation of the Rad53 kinase. Rad53 is essential in maintaining cell viability, but its targets of regulation are still partially unknown. Here we show that Rad53 drives the hyper-SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε, principally following replication forks stalling induced by nucleotide depletion. Pol2 is the main target of SUMOylation within the replisome and its modification requires the SUMO-ligase Mms21, a subunit of the Smc5/6 complex. Moreover, the Smc5/6 complex co-purifies with Pol ε, independently of other replisome components. Finally, we map Pol2 SUMOylation to a single site within the N-terminal catalytic domain and identify a SUMO-interacting motif at the C-terminus of Pol2. These data suggest that the S phase checkpoint regulate Pol ε during replication stress through Pol2 SUMOylation and SUMO-binding abilit

Damage of DNA and proteins by major lipid peroxidation products in genome stability

Oxidative stress and lipid peroxidation (LPO) accompanying infections and chronic inflammation may induce several human cancers. LPO products are characterized by carbohydrate chains of different length, reactive aldehyde groups and double bonds, which make these molecules reactive to nucleic acids, proteins and cellular thiols. LPO-derived adducts to DNA bases form etheno-type and propano-type exocyclic rings, which have profound mutagenic potential, and are elevated in several cancer-prone diseases. Adducts of long chain LPO products to DNA bases inhibits transcription. Elimination from DNA of LPO-induced lesions is executed by several repair systems: base excision repair (BER), direct reversal by AlkB family proteins, nucleotide excision repair (NER) and recombination. Modifications of proteins with LPO products may regulate cellular processes like apoptosis, cell signaling and senescence. This review summarizes consequences of LPO products presence in cell, particularly 4-hydroxy-2-nonenal in terms of genomic stability

Lipid peroxidation product 4-hydroxy-2-nonenal modulates base excision repair in human cells

International audienc