Mind the replication gap

Unlike bacteria, mammalian cells need to complete DNA replication before segregating their chromosomes for the maintenance of genome integrity. Thus, cells have evolved efficient pathways to restore stalled and/or collapsed replication forks during S-phase, and when necessary, also to delay cell cycle progression to ensure replication completion. However, strong evidence shows that cells can proceed to mitosis with incompletely replicated DNA when under mild replication stress (RS) conditions. Consequently, the incompletely replicated genomic gaps form, predominantly at common fragile site regions, where the converging fork-like DNA structures accumulate. These branched structures pose a severe threat to the faithful disjunction of chromosomes as they physically interlink the partially duplicated sister chromatids. In this review, we provide an overview discussing how cells respond and deal with the under-replicated DNA structures that escape from the S/G2 surveillance system. We also focus on recent research of a mitotic break-induced replication pathway (also known as mitotic DNA repair synthesis), which has been proposed to operate during prophase in an attempt to finish DNA synthesis at the under-replicated genomic regions. Finally, we discuss recent data on how mild RS may cause chromosome instability and mutations that accelerate cancer genome evolution

The unresolved problem of DNA bridging

Accurate duplication and transmission of identical genetic information into offspring cells lies at the heart of a cell division cycle. During the last stage of cellular division, namely mitosis, the fully replicated DNA molecules are condensed into X-shaped chromosomes, followed by a chromosome separation process called sister chromatid disjunction. This process allows for the equal partition of genetic material into two newly born daughter cells. However, emerging evidence has shown that faithful chromosome segregation is challenged by the presence of persistent DNA intertwining structures generated during DNA replication and repair, which manifest as so-called ultra-fine DNA bridges (UFBs) during anaphase. Undoubtedly, failure to disentangle DNA linkages poses a severe threat to mitosis and genome integrity. This review will summarize the possible causes of DNA bridges, particularly sister DNA inter-linkage structures, in an attempt to explain how they may be processed and how they influence faithful chromosome segregation and the maintenance of genome stability

Another string to the polo bow: a new mitotic role of PLK1 in centromere protection

Polo-like kinase 1 (PLK1) plays a fundamental role in the spatiotemporal control of mitosis. Cells lacking PLK1 activity exhibit characteristic chromosome misalignment due to defects in microtubule-kinetochore organization and attachment. In our recently published paper, we uncover a new role for PLK1 in the preservation and maintenance of centromere integrity

Biological chromodynamics: a general method for measuring protein occupancy across the genome by calibrating ChIP-seq

Sequencing DNA fragments associated with proteins following in vivo cross-linking with formaldehyde (known as ChIP-seq) has been used extensively to describe the distribution of proteins across genomes. It is not widely appreciated that this method merely estimates a protein's distribution and cannot reveal changes in occupancy between samples. To do this, we tagged with the same epitope orthologous proteins in Saccharomyces cerevisiae and Candida glabrata, whose sequences have diverged to a degree that most DNA fragments longer than 50 bp are unique to just one species. By mixing defined numbers of C. glabrata cells (the calibration genome) with S. cerevisiae samples (the experimental genomes) prior to chromatin fragmentation and immunoprecipitation, it is possible to derive a quantitative measure of occupancy (the occupancy ratio – OR) that enables a comparison of occupancies not only within but also between genomes. We demonstrate for the first time that this ‘internal standard’ calibration method satisfies the sine qua non for quantifying ChIP-seq profiles, namely linearity over a wide range. Crucially, by employing functional tagged proteins, our calibration process describes a method that distinguishes genuine association within ChIP-seq profiles from background noise. Our method is applicable to any protein, not merely highly conserved ones, and obviates the need for the time consuming, expensive, and technically demanding quantification of ChIP using qPCR, which can only be performed on individual loci. As we demonstrate for the first time in this paper, calibrated ChIP-seq represents a major step towards documenting the quantitative distributions of proteins along chromosomes in different cell states, which we term biological chromodynamics

DNA replication is highly resilient and persistent under the challenge of mild replication stress

Mitotic DNA synthesis (MiDAS) has been proposed to restart DNA synthesis during mitosis because of replication fork stalling in late interphase caused by mild replication stress (RS). Contrary to this proposal, we find that cells exposed to mild RS in fact maintain continued DNA replication throughout G2 and during G2-M transition in RAD51- and RAD52-dependent manners. Persistent DNA synthesis is necessary to resolve replication intermediates accumulated in G2 and disengage an ATR-imposed block to mitotic entry. Because of its continual nature, DNA synthesis at very late replication sites can overlap with chromosome condensation, generating the phenomenon of mitotic DNA synthesis. Unexpectedly, we find that the commonly used CDK1 inhibitor RO3306 interferes with replication to preclude detection of G2 DNA synthesis, leading to the impression of a mitosis-driven response. Our study reveals the importance of persistent DNA replication and checkpoint control to lessen the risk for severe genome under-replication under mild RS

Releasing activity disengages Cohesin’s Smc3/Scc1 interface in a process blocked by Acetylation

Sister chromatid cohesion conferred by entrapment of sister DNAs within a tripartite ring formed between cohesin’s Scc1, Smc1, and Smc3 subunits is created during S and destroyed at anaphase through Scc1 cleavage by separase. Cohesin’s association with chromosomes is controlled by opposing activities: loading by Scc2/4 complex and release by a separase- independent releasing activity as well as by cleavage. Coentrapment of sister DNAs at replication is accompanied by acetylation of Smc3 by Eco1, which blocks releasing activity and ensures that sisters remain connected. Because fusion of Smc3 to Scc1 prevents release and bypasses the requirement for Eco1, we suggested that release is mediated by disengagement of the Smc3/Scc1 interface. We show that mutations capable of bypassing Eco1 in Smc1, Smc3, Scc1, Wapl, Pds5, and Scc3 subunits reduce dissociation of N-terminal cleavage fragments of Scc1 (NScc1) from Smc3. This process involves interaction between Smc ATPase heads and is inhibited by Smc3 acetylation

The cohesin ring uses its hinge to organize DNA using non-topological as well as topological mechanisms

As predicted by the notion that sister chromatid cohesion is mediated by entrapment of sister DNAs inside cohesin rings, there is perfect correlation between co-entrapment of circular minichromosomes and sister chromatid cohesion. In most cells where cohesin loads without conferring cohesion, it does so by entrapment of individual DNAs. However, cohesin with a hinge domain whose positively charged lumen is neutralized loads and moves along chromatin despite failing to entrap DNAs. Thus, cohesin engages chromatin in non-topological, as well as topological, manners. Since hinge mutations, but not Smc-kleisin fusions, abolish entrapment, DNAs may enter cohesin rings through hinge opening. Mutation of three highly conserved lysine residues inside the Smc1 moiety of Smc1/3 hinges abolishes all loading without affecting cohesin’s recruitment to CEN loading sites or its ability to hydrolyze ATP. We suggest that loading and translocation are mediated by conformational changes in cohesin’s hinge driven by cycles of ATP hydrolysis

FIRRM/C1orf112 is synthetic lethal with PICH and mediates RAD51 dynamics

Joint DNA molecules are natural byproducts of DNA replication and repair. Persistent joint molecules give rise to ultrafine DNA bridges (UFBs) in mitosis, compromising sister chromatid separation. The DNA translocase PICH (ERCC6L) has a central role in UFB resolution. A genome-wide loss-of-function screen is performed to identify the genetic context of PICH dependency. In addition to genes involved in DNA condensation, centromere stability, and DNA-damage repair, we identify FIGNL1-interacting regulator of recombination and mitosis (FIRRM), formerly known as C1orf112. We find that FIRRM interacts with and stabilizes the AAA + ATPase FIGNL1. Inactivation of either FIRRM or FIGNL1 results in UFB formation, prolonged accumulation of RAD51 at nuclear foci, and impaired replication fork dynamics and consequently impairs genome maintenance. Combined, our data suggest that inactivation of FIRRM and FIGNL1 dysregulates RAD51 dynamics at replication forks, resulting in persistent DNA lesions and a dependency on PICH to preserve cell viability. </p

Roles of the Bloom's syndrome helicase in the maintenance of chromosome stability

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