The p97 cofactor Ubxn7 facilitates replisome disassembly during S-phase

Complex cellular processes are driven by the regulated assembly and disassembly of large multi-protein complexes. In eukaryotic DNA replication, whilst we are beginning to understand the molecular mechanism for assembly of the replication machinery (replisome), we still know relatively little about the regulation of its disassembly at replication termination. Over recent years, the first elements of this process have emerged, revealing that the replicative helicase, at the heart of the replisome, is polyubiquitylated prior to unloading and that this unloading requires p97 segregase activity. Two different E3 ubiquitin ligases are now known to ubiquitylate the helicase under different conditions: Cul2Lrr1 and TRAIP. Here we have found two p97 cofactors, Ubxn7 and Faf1, which can interact with p97 during replisome disassembly in S-phase. Only Ubxn7 however facilitates efficient replisome disassembly through its interaction with both Cul2Lrr1 and p97. Our data therefore characterise Ubxn7 as the first substrate-specific p97 cofactor regulating replisome disassembly in vertebrates.

Genomic analysis finds no evidence of canonical eukaryotic DNA processing complexes in a free-living protist

Cells replicate and segregate their DNA with precision. Previous studies showed that these regulated cell-cycle processes were present in the last eukaryotic common ancestor and that their core molecular parts are conserved across eukaryotes. However, some metamonad parasites have secondarily lost components of the DNA processing and segregation apparatuses. To clarify the evolutionary history of these systems in these unusual eukaryotes, we generated a genome assembly for the free-living metamonad Carpediemonas membranifera and carried out a comparative genomics analysis. Here, we show that parasitic and free-living metamonads harbor an incomplete set of proteins for processing and segregating DNA. Unexpectedly, Carpediemonas species are further streamlined, lacking the origin recognition complex, Cdc6 and most structural kinetochore subunits. Carpediemonas species are thus the first known eukaryotes that appear to lack this suite of conserved complexes, suggesting that they likely rely on yet-to-be-discovered or alternative mechanisms to carry out these fundamental processes.

Variations in Nucleosome Structure and Organization among Eukaryotes

Folding eukaryotic DNA by chromatin is a vital process necessary for the proper function of DNA. This is achieved by the fundamental unit of chromatin, known as a nucleosome. The position of a nucleosome and its interaction with DNA plays a crucial role in regulating the vital processes involved in DNA function. Factors such as variations in nucleosome and its core structure and histone fold variations will help to understand nucleosome functions and their role in DNA replication, transcription, translation, posttranslational modifications, re-combinations and repair. The present review focuses on recent findings in understanding the variations in the structure and functions of nucleosomes across eukaryotes. Variations in the nucleosome organization and its assembly have also been discussed by stating the contribution of histone binding factors and chromatin assembly factors.

Rad27 and Exo1 function in different excision pathways for mismatch repair in Saccharomyces cerevisiae

Eukaryotic DNA Mismatch Repair (MMR) involves redundant exonuclease 1 (Exo1)-dependent and Exo1-independent pathways, of which the Exo1-independent pathway(s) is not well understood. The exo1Δ440-702 mutation, which deletes the MutS Homolog 2 (Msh2) and MutL Homolog 1 (Mlh1) interacting peptides (SHIP and MIP boxes, respectively), eliminates the Exo1 MMR functions but is not lethal in combination with rad27Δ mutations. Analyzing the effect of different combinations of the exo1Δ440-702 mutation, a rad27Δ mutation and the pms1-A99V mutation, which inactivates an Exo1-independent MMR pathway, demonstrated that each of these mutations inactivates a different MMR pathway. Furthermore, it was possible to reconstitute a Rad27- and Msh2-Msh6-dependent MMR reaction in vitro using a mispaired DNA substrate and other MMR proteins. Our results demonstrate Rad27 defines an Exo1-independent eukaryotic MMR pathway that is redundant with at least two other MMR pathways.

The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.

The Alterations and Potential Roles of MCMs in Breast Cancer

The minichromosome maintenance (MCM) protein family plays a key role in eukaryotic DNA replication and has been confirmed to be associated with the occurrence and progression of many tumors. However, the expression levels, functions, and prognostic values of MCMs in breast cancer (BC) have not been clearly and systematically explained. In this article, we studied the transcriptional levels of MCMs in BC based on the Oncomine database. Kaplan-Meier plotter was used to analyze prognostic value of MCMs in human BC patients. Furthermore, we constructed a MCM coexpression gene network and performed functional annotation analysis through DAVID to reveal the functions of MCMs and coexpressed genes. The data showed that the expression of MCM2–8 and MCM10 but not MCM1 and MCM9 was upregulated in BC. Kaplan-Meier plotter analysis revealed that high transcriptional levels of MCM2, MCM4–7, and MCM10 were significantly related to low relapse-free survival (RFS) in BC patients. In contrast, high levels of MCM1 and MCM9 predicted high RFS for BC patients. This study suggests that MCM2, MCM4–7, and MCM10 possess great potential to be valuable prognostic biomarkers for BC and that MCM1 and MCM9 may serve as potential treatment targets for BC patients.

ORC binds and remodels nucleosomes to specify MCM loading onto DNA

ABSTRACTSaccharomyces cerevisiae has been a faithful guide for study of eukaryotic DNA replication, as the numerous initiation and elongation proteins are conserved from yeast to human. However, there is a gap in our knowledge of why yeast uses a consensus DNA sequence at replication origins, while higher eukaryotes do not. The current study closes this gap. By direct single-molecule visualization, we show that the Origin Recognition Complex (ORC) searches for and stably binds nucleosomes, and that nucleosomes funtionalize ORC to load MCM helicase onto DNA, regardless of DNA sequence. Furthermore, we discover that ORC can remodel nucleosomes and expel H2A-H2B histone dimers, a heretofore unexpected function. Thus ORC helps create a chromatin environment permissive to origin function. The finding that ORC binding to nucleosomes leads to MCM loading at any DNA sequence is likely to generalize, and that higher eukaryotes follow this same paradigm for origin selection

Bacterial N4-methylcytosine as an epigenetic mark in eukaryotic DNA

In eukaryotes, 5-methylcytosine is the predominant DNA base modification, followed by N6-methyladenine. However, N4-methylcytosine (4mC) is confined to bacteria. Here we report that 4mC can serve as an epigenetic mark in eukaryotes. Bdelloid rotifers, freshwater invertebrates with transposon-poor genomes that are rich in foreign genes, lack C5-methyltransferases but encode an amino-methyltransferase, N4CMT, captured from bacteria >60 Mya. N4CMT introduces 4mC into DNA, and its chromodomain shapes the "histone-read-DNA-write" architecture together with a "DNA-read-histone-write" SETDB1/eggless H3K9me3 histone methyltransferase variant preferentially binding 4mC-DNA, to maintain 4mC and silent chromatin at transposons and tandem repeats. Our results bring the third base modification into the eukaryotic repertoire, demonstrate how non-native DNA methyl groups can reshape complex epigenetic systems to suppress transposon proliferation, and establish horizontal gene transfer as the source of regulatory innovation in eukaryotes.

The interplay of RNA:DNA hybrid structure and G-quadruplexes determines the outcome of R-loop-replisome collisions

R-loops are a major source of genome instability associated with transcription-induced replication stress. However, how R-loops inherently impact replication fork progression is not understood. Here, we characterize R-loop-replisome collisions using a fully reconstituted eukaryotic DNA replication system. We find that RNA:DNA hybrids and G-quadruplexes at both co-directional and head-on R-loops can impact fork progression by inducing fork stalling, uncoupling of leading strand synthesis from replisome progression, and nascent strand gaps. RNase H1 and Pif1 suppress replication defects by resolving RNA:DNA hybrids and G-quadruplexes, respectively. We also identify an intrinsic capacity of replisomes to maintain fork progression at certain R-loops by unwinding RNA:DNA hybrids, repriming leading strand synthesis downstream of G-quadruplexes, or utilizing R-loop transcripts to prime leading strand restart during co-directional R-loop-replisome collisions. Collectively, the data demonstrates that the outcome of R-loop-replisome collisions is modulated by R-loop structure, providing a mechanistic basis for the distinction of deleterious from non-deleterious R-loops.