Mitotic DNA Synthesis in Untransformed Human Cells Preserves Common Fragile Site Stability via a FANCD2-Driven Mechanism That Requires HELQ.

Faithful genome duplication is a challenging task for dividing mammalian cells, particularly under replication stress where timely resolution of late replication intermediates (LRIs) becomes crucial prior to cell division. In human cancer cells, mitotic DNA repair synthesis (MiDAS) is described as a final mechanism for the resolution of LRIs to avoid lethal chromosome mis-segregation. RAD52-driven MiDAS achieves this mission in part by generating gaps/breaks on metaphase chromosomes, which preferentially occur at common fragile sites (CFS).

Walking Attainment in Very Low Birth Weight Infants in Japan.

Objective: To clarify the corrected age of walking attainment in very low birth weight infants by birth weight and gestational age, and determine perinatal factors affecting the delay in walking attainment.

Method: This was a longitudinal study. We investigated walking attainment and perinatal factors in 145 very low birth weight infants without neurological abnormalities (mean birth weight 1019.

Staged Bilateral Total Knee Arthroplasty in a Patient with Charcot Knees: A Case Report.

Introduction: Charcot arthropathy causes severe progressive and destructive joint disease. With the development of prostheses and surgical techniques, orthopedic surgeons have a greater opportunity to use total knee arthroplasty (TKA) to treat Charcot knee. However, consensus is lacking regarding prosthesis choice.

Effects of continued positioning pillow use until a corrected age of six months on cranial deformation and neurodevelopment in preterm infants: A prospective case-control study.

Background: Preterm infants have a high risk of cranial deformity resulting from external pressures. Such deformity is associated with delayed neurodevelopment.

Aims: We aimed to clarify the effects of continuous use of positioning pillows on cranial deformity and neurodevelopment in preterm infants.

Mitotic DNA Synthesis Is Differentially Regulated between Cancer and Noncancerous Cells.

Mitotic DNA synthesis is a recently discovered mechanism that resolves late replication intermediates, thereby supporting cell proliferation under replication stress. This unusual form of DNA synthesis occurs in the absence of RAD51 or BRCA2, which led to the identification of RAD52 as a key player in this process. Notably, mitotic DNA synthesis is predominantly observed at chromosome loci that colocalize with FANCD2 foci.

Dormant origins as a built-in safeguard in eukaryotic DNA replication against genome instability and disease development.

DNA replication is a prerequisite for cell proliferation, yet it can be increasingly challenging for a eukaryotic cell to faithfully duplicate its genome as its size and complexity expands. Dormant origins now emerge as a key component for cells to successfully accomplish such a demanding but essential task. In this perspective, we will first provide an overview of the fundamental processes eukaryotic cells have developed to regulate origin licensing and firing.

Embryonic Stem Cells License a High Level of Dormant Origins to Protect the Genome against Replication Stress.

Maintaining genomic integrity during DNA replication is essential for stem cells. DNA replication origins are licensed by the MCM2-7 complexes, with most of them remaining dormant. Dormant origins (DOs) rescue replication fork stalling in S phase and ensure genome integrity.

Methods for the detection of genome instability derived from replication stress in primary mouse embryonic fibroblasts.

Replication stress, with its subsequent genome instability, is a hallmark of cancer from its earliest stages of development. Here, we describe assays that are sufficiently sensitive to detect intrinsic replicative stress and its consequences in primary mouse embryonic fibroblasts. First, we explain the non-denatured DNA fiber assay, a powerful tool to directly measure DNA replication kinetics via the dual-labeling of active replication forks.

Hypersensitivity of primordial germ cells to compromised replication-associated DNA repair involves ATM-p53-p21 signaling.

Genome maintenance in germ cells is critical for fertility and the stable propagation of species. While mechanisms of meiotic DNA repair and chromosome behavior are well-characterized, the same is not true for primordial germ cells (PGCs), which arise and propagate during very early stages of mammalian development. Fanconi anemia (FA), a genomic instability syndrome that includes hypogonadism and testicular failure phenotypes, is caused by mutations in genes encoding a complex of proteins involved in repair of DNA lesions associated with DNA replication.

A concomitant loss of dormant origins and FANCC exacerbates genome instability by impairing DNA replication fork progression.

Accumulating evidence suggests that dormant DNA replication origins play an important role in the recovery of stalled forks. However, their functional interactions with other fork recovery mechanisms have not been tested. We previously reported intrinsic activation of the Fanconi anemia (FA) pathway in a tumor-prone mouse model (Mcm4chaos3) with a 60% loss of dormant origins.

Helq acts in parallel to Fancc to suppress replication-associated genome instability.

HELQ is a superfamily 2 DNA helicase found in archaea and metazoans. It has been implicated in processing stalled replication forks and in repairing DNA double-strand breaks and inter-strand crosslinks. Though previous studies have suggested the possibility that HELQ is involved in the Fanconi anemia (FA) pathway, a dominant mechanism for inter-strand crosslink repair in vertebrates, this connection remains elusive.

A reduction of licensed origins reveals strain-specific replication dynamics in mice.

Replication origin licensing builds a fundamental basis for DNA replication in all eukaryotes. This occurs during the late M to early G1 phases in which chromatin is licensed by loading of the MCM2-7 complex, an essential component of the replicative helicase. In the following S phase, only a minor fraction of chromatin-bound MCM2-7 complexes are activated to unwind the DNA.

Stalled fork rescue via dormant replication origins in unchallenged S phase promotes proper chromosome segregation and tumor suppression.

Eukaryotic cells license far more origins than are actually used for DNA replication, thereby generating a large number of dormant origins. Accumulating evidence suggests that such origins play a role in chromosome stability and tumor suppression, though the underlying mechanism is largely unknown. Here, we show that a loss of dormant origins results in an increased number of stalled replication forks, even in unchallenged S phase in primary mouse fibroblasts derived from embryos homozygous for the Mcm4(Chaos3) allele.

Functional screen of human MCM2-7 variant alleles for disease-causing potential.

Origin licensing builds a fundamental basis for genome stability in DNA replication. Recent studies reported that deregulation of origin licensing is associated with replication stress in precancerous lesions. The heterohexameric complex of minichromosome maintenance proteins (MCM2-7 complex) plays an essential role in origin licensing.

Genetic screen for chromosome instability in mice: Mcm4 and breast cancer.

We recently isolated a hypomorphic mutation of Mcm4 in a phenotype-based screen for chromosome instability in mice. This mutation, named Chaos3 (chromosome aberrations occurring spontaneously 3), causes exclusively mammary adenocarcinomas in approximately 80% of homozygous females. Mcm4 encodes a subunit of the MCM2-7 complex, the replication-licensing factor and the replicative helicase.

A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice.

Mcm4 (minichromosome maintenance-deficient 4 homolog) encodes a subunit of the MCM2-7 complex (also known as MCM2-MCM7), the replication licensing factor and presumptive replicative helicase. Here, we report that the mouse chromosome instability mutation Chaos3 (chromosome aberrations occurring spontaneously 3), isolated in a forward genetic screen, is a viable allele of Mcm4. Mcm4(Chaos3) encodes a change in an evolutionarily invariant amino acid (F345I), producing an apparently destabilized MCM4.

The translesion DNA polymerase theta plays a dominant role in immunoglobulin gene somatic hypermutation.

Immunoglobulin (Ig) somatic hypermutation (SHM) critically underlies the generation of high-affinity antibodies. Mutations can be introduced by error-prone polymerases such as polymerase zeta (Rev3), a mispair extender, and polymerase eta, a mispair inserter with a preference for dA/dT, while repairing DNA lesions initiated by AID-mediated deamination of dC to yield dU:dG mismatches. The partial impairment of SHM observed in the absence of these polymerases led us to hypothesize a main role for another translesion DNA polymerase.

The mouse genomic instability mutation chaos1 is an allele of Polq that exhibits genetic interaction with Atm.

chaos1 (for chromosome aberrations occurring spontaneously 1) is a recessive mutation that was originally identified in a phenotype-based screen for chromosome instability mutants in mice. Mutant animals exhibit significantly higher frequencies of spontaneous and radiation- or mitomycin C-induced micronucleated erythrocytes, indicating a potential defect in homologous recombination or interstrand cross-link repair. The chaos1 allele was genetically associated with a missense mutation in Polq, which encodes DNA polymerase theta;.

Forward genetic screens for meiotic and mitotic recombination-defective mutants in mice.

The goal of understanding the function of all mammalian genes is best accomplished through mutational analyses. Although the sequence of the mouse genome is now available and many genes have been identified, it is not possible to ascribe functions accurately to these genes in silico. Gene targeting using embryonic stem cells is ideal for analysis of individual genes selected on the basis of sequence features, but it is impractical for identifying novel genes involved in particular biological processes.

Distribution of estrogen receptor beta mRNA-containing cells in ovariectomized and estrogen-treated female rat brain.

Estrogen receptor (ER)-beta is a member of the nuclear receptor superfamily and mediates various estrogenic actions. Changes in ER-alpha mRNA expression induced by estrogen have been well documented, whereas those with regard to ER-beta have only been reported for a part of the hypothalamus. In the present study, we examined the effect of estrogen on ER-beta mRNA expression in the female rat brain.

Phenotype-based identification of mouse chromosome instability mutants.

There is increasing evidence that defects in DNA double-strand-break (DSB) repair can cause chromosome instability, which may result in cancer. To identify novel DSB repair genes in mice, we performed a phenotype-driven mutagenesis screen for chromosome instability mutants using a flow cytometric peripheral blood micronucleus assay. Micronucleus levels were used as a quantitative indicator of chromosome damage in vivo.