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.

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.

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.

PLK1 facilitates chromosome biorientation by suppressing centromere disintegration driven by BLM-mediated unwinding and spindle pulling.

Centromeres provide a pivotal function for faithful chromosome segregation. They serve as a foundation for the assembly of the kinetochore complex and spindle connection, which is essential for chromosome biorientation. Cells lacking Polo-like kinase 1 (PLK1) activity suffer severe chromosome alignment defects, which is believed primarily due to unstable kinetochore-microtubule attachment.

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.

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.

53BP1 can limit sister-chromatid rupture and rearrangements driven by a distinct ultrafine DNA bridging-breakage process.

Chromosome missegregation acts as one of the driving forces for chromosome instability and cancer development. Here, we find that in human cancer cells, HeLa and U2OS, depletion of 53BP1 (p53-binding protein 1) exacerbates chromosome non-disjunction resulting from a new type of sister-chromatid intertwinement, which is distinct from FANCD2-associated ultrafine DNA bridges (UFBs) induced by replication stress. Importantly, the sister DNA intertwinements trigger gross chromosomal rearrangements through a distinct process, named sister-chromatid rupture and bridging.

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.

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.

Closing the cohesin ring: structure and function of its Smc3-kleisin interface.

Through their association with a kleisin subunit (Scc1), cohesin's Smc1 and Smc3 subunits are thought to form tripartite rings that mediate sister chromatid cohesion. Unlike the structure of Smc1/Smc3 and Smc1/Scc1 interfaces, that of Smc3/Scc1 is not known. Disconnection of this interface is thought to release cohesin from chromosomes in a process regulated by acetylation.

Structure and function of cohesin's Scc3/SA regulatory subunit.

Sister chromatid cohesion involves entrapment of sister DNAs by a cohesin ring created through association of a kleisin subunit (Scc1) with ATPase heads of Smc1/Smc3 heterodimers. Cohesin's association with chromatin involves subunits recruited by Scc1: Wapl, Pds5, and Scc3/SA, in addition to Scc2/4 loading complex. Unlike Pds5, Wapl, and Scc2/4, Scc3s are encoded by all eukaryotic genomes.

Pds5 promotes and protects cohesin acetylation.

Cohesin's Smc1 and Smc3 subunits form V-shaped heterodimers, the nucleotide binding domains (NBDs) of which bind the C- and N-terminal domains, respectively, of the α-kleisin subunit, forming a large tripartite ring within in which sister DNAs are entrapped, and thereby held together (sister chromatid cohesion). During replication, establishment of stable cohesion is dependent on Eco1-mediated acetylation of Smc3's NBD, which is thought to prevent dissociation of α-kleisin from Smc3, thereby locking shut a "DNA exit gate." How Scc3 and Pds5, regulatory subunits bound to α-kleisin, regulate cohesion establishment and maintenance is poorly understood.

MUS81 promotes common fragile site expression.

Fragile sites are chromosomal loci with a propensity to form gaps or breaks during early mitosis, and their instability is implicated as being causative in certain neurological disorders and cancers. Recent work has demonstrated that the so-called common fragile sites (CFSs) often impair the faithful disjunction of sister chromatids in mitosis. However, the mechanisms by which CFSs express their fragility, and the cellular factors required to suppress CFS instability, remain largely undefined.

Cohesin's DNA exit gate is distinct from its entrance gate and is regulated by acetylation.

Sister chromatid cohesion is mediated by entrapment of sister DNAs by a tripartite ring composed of cohesin's Smc1, Smc3, and α-kleisin subunits. Cohesion requires acetylation of Smc3 by Eco1, whose role is to counteract an inhibitory (antiestablishment) activity associated with cohesin's Wapl subunit. We show that mutations abrogating antiestablishment activity also reduce turnover of cohesin on pericentric chromatin.

New insights into the formation and resolution of ultra-fine anaphase bridges.

Recent data indicate an unexpected requirement for proteins that were hitherto considered to be dedicated to DNA repair to facilitate the faithful disjunction of sister chromatids in anaphase. These include the Bloom's syndrome gene product, BLM and its partners, as well as a number of proteins that are important for preventing Fanconi anemia (FA) in man. As part of an analysis of the roles of these proteins in mitosis, we identified a novel class of anaphase bridge structure, called an ultra-fine anaphase bridge (UFB).

53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress.

Completion of genome duplication is challenged by structural and topological barriers that impede progression of replication forks. Although this can seriously undermine genome integrity, the fate of DNA with unresolved replication intermediates is not known. Here, we show that mild replication stress increases the frequency of chromosomal lesions that are transmitted to daughter cells.

ATP hydrolysis is required for relocating cohesin from sites occupied by its Scc2/4 loading complex.

Background: The Cohesin complex that holds sister chromatins together until anaphase is comprised of three core subunits: Smc1 and Smc3, two long-rod-shaped proteins with an ABC-like ATPase head (nucleotide-binding domain [NBD]) and a dimerization domain linked by a 50 nm long intramolecular antiparallel coiled-coil, and Scc1, an α-kleisin subunit interconnecting the NBD domains of Smc1 and Smc3. Cohesin's stable association with chromosomes is thought to involve entrapment of chromatin fibers by its tripartite Smc1-Smc3-Scc1 ring via a poorly understood mechanism dependent on a separate Scc2/4 loading complex. A key issue concerns where entrapment initially takes place: at sites where cohesin is found stably associated or at distinct "loading" sites from which it translocates.

Replication stress induces sister-chromatid bridging at fragile site loci in mitosis.

Several inherited syndromes in humans are associated with cancer predisposition. The gene products defective in two of these disorders, BLM (a helicase defective in Bloom's syndrome) and FANC A-N (defective in Fanconi anaemia), associate in a multienzyme complex called BRAFT. How these proteins suppress tumorigenesis remains unclear, although both conditions are associated with chromosome instability.

BLM is required for faithful chromosome segregation and its localization defines a class of ultrafine anaphase bridges.

Mutations in BLM cause Bloom's syndrome, a disorder associated with cancer predisposition and chromosomal instability. We investigated whether BLM plays a role in ensuring the faithful chromosome segregation in human cells. We show that BLM-defective cells display a higher frequency of anaphase bridges and lagging chromatin than do isogenic corrected derivatives that eptopically express the BLM protein.

The HRDC domain of BLM is required for the dissolution of double Holliday junctions.

Bloom's syndrome is a hereditary cancer-predisposition disorder resulting from mutations in the BLM gene. In humans, BLM encodes one of five members of the RecQ helicase family. One function of BLM is to act in concert with topoisomerase IIIalpha (TOPO IIIalpha) to resolve recombination intermediates containing double Holliday junctions by a process called double Holliday junction dissolution, herein termed dissolution.

High-throughput tissue microarray analysis of c-myc activation in chronic liver diseases and hepatocellular carcinoma.

Amplification of 8q23-qter is common in human hepatocellular carcinoma (HCC). c-myc, an oncogene located on 8q24, may be important in hepatocarcinogenesis. The present study aimed to evaluate c-myc activation in hepatocarcinogenesis and its clinicopathological significance.

HDPR1, a novel inhibitor of the WNT/beta-catenin signaling, is frequently downregulated in hepatocellular carcinoma: involvement of methylation-mediated gene silencing.

Oncogenic activation of the WNT/beta-catenin signaling pathway is common in hepatocellular carcinoma (HCC). Dishevelled (Dvl), a key activator of the pathway, inhibits the adenomatous polyposis coli complex, and this leads to the accumulation of beta-catenin and promotes tumorigenesis. Recently, a novel inhibitor of Dishevelled, namely Dapper (Dpr), was isolated in Xenopus.

High frequency of chimerism in transplanted livers.

Recent studies have shown that primitive stem cells can mobilize and differentiate into hepatocytes. We investigated the time and extent in which cells of recipient origins could differentiate into hepatocytes and other cells in human liver allografts. Microsatellite analysis, which can assess quantitatively the proportions of recipient and donor DNA, was performed in posttransplantation liver biopsy specimens from 17 patients at various times.