Publications by authors named "Naoko Shima"

19 Publications

  • Page 1 of 1

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

J Orthop Case Rep 2020 ;10(2):9-12

Department of Orthopedic Surgery, Hyogo Prefectural Central Rehabilitation Hospital,1070 Akebono-chou, Nishi-ku, Kobe, Hyogo, 651-2181 Japan.

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. Here, we present a case of staged bilateral TKA in a patient with bilateral Charcot knees in a different way.

Case Report: We report a case of a 64-year-old woman with bilateral Charcot knees. Her knee joints had become increasingly unstable with severe deformity over 1 year, and she has been unable to walk for1 month before hospitalization. We performed the first TKA of the right knee using rotating hinge prosthesis with a combination of autologous bone graft and metal tibial block augmentation; lateral release and patellar replacement were also performed. Three months after the first surgery, we performed the second TKA for her left knee using a constrained condylar prosthesis with a combination of lateral retinaculum release and patella replacement. At the 6-month follow-up, both knees were stable and in good alignment. There were no signs of loosening or fracture. The patient experienced no pain and was able to ambulate in her household using a walker.

Conclusion: TKA using constrained condylar or rotating hinge prosthesis effectively treats Charcot knee. Surgeons must acquire both appropriate alignment and ligament stability rather than the range of motion to ensure increased longevity of the prosthesis by performing various surgical techniques. Careful follow-up is needed, but Charcot knee patients can have good outcomes with TKA.
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http://dx.doi.org/10.13107/jocr.2020.v10.i02.1674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476687PMC
January 2020

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.

Early Hum Dev 2020 09 9;148:105137. Epub 2020 Jul 9.

Department of Physical Therapy, Graduate School of Human Health Sciences, Tokyo Metropolitan University, Tokyo, Japan.

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.

Methods: This prospective case-control study was conducted between November 2018 and August 2019. The continuous use of a pillow was initiated after discharge from the neonatal intensive care unit, up to a corrected age of six months. Preterm infants weighing less than 1800 g without neurological abnormalities were included in the study. Patients were divided into two groups: non-pillow group (NP-group) and pillow group (P-group). The primary outcome was the Bayley Scales of Infant Development III (BSID-III) score. We compared asymmetrical cranial deformity and the BSID-III scores in the two groups at a corrected age of six months using the Fisher's exact test and unpaired t-test, respectively.

Results: There were 19 preterm infants (mean gestational age 32.5 ± 1.9 weeks, birth weight 1461.3 ± 244.7) eligible during the study period. The P-group (n = 11) showed asymmetrical cranial deformity at six months less frequently than the NP-group (n = 8) (p = 0.001, Fisher's exact test). Infants in the P-group had significantly higher scores on the BSID-III cognitive scales (95.0 ± 8.4 vs. 86.9 ± 2.6; p = 0.02, unpaired t-test) and fine motor scores on the motor scales (8.6 ± 2.2 vs. 6.6 ± 0.7, p = 0.02, unpaired t-test).

Conclusions: Continuous pillow use in preterm infants is effective in reducing cranial deformity and improved cognitive and fine motor skills.

Trial Registration: UMIN Clinical Trials Registry, trial no. UMIN000034400 (http://www.umin.ac.jp/ctr/index.htm).
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http://dx.doi.org/10.1016/j.earlhumdev.2020.105137DOI Listing
September 2020

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

Mol Cancer Res 2019 08 21;17(8):1687-1698. Epub 2019 May 21.

Department of Genetics, Cell Biology and Development, University of Minnesota, at Twin Cities, Masonic Cancer Center, Minneapolis, Minnesota.

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. However, the role of this protein in mitotic DNA synthesis remains largely unknown. In this study, we investigated the role of FANCD2 and its interplay with RAD52 in mitotic DNA synthesis using aphidicolin as a universal inducer of this process. After examining eight human cell lines, we provide evidence for FANCD2 rather than RAD52 as a fundamental supporter of mitotic DNA synthesis. In cancer cell lines, FANCD2 exerts this role independently of RAD52. Surprisingly, RAD52 is dispensable for mitotic DNA synthesis in noncancerous cell lines, but these cells strongly depend on FANCD2 for this process. Therefore, RAD52 functions selectively in cancer cells as a secondary regulator in addition to FANCD2 to facilitate mitotic DNA synthesis. As an alternative to aphidicolin, we found partial inhibition of origin licensing as an effective way to induce mitotic DNA synthesis preferentially in cancer cells. Importantly, cancer cells still perform mitotic DNA synthesis by dual regulation of FANCD2 and RAD52 under such conditions. IMPLICATIONS: These key differences in mitotic DNA synthesis between cancer and noncancerous cells advance our understanding of this mechanism and can be exploited for cancer therapies.
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http://dx.doi.org/10.1158/1541-7786.MCR-19-0057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6677588PMC
August 2019

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

DNA Repair (Amst) 2017 08 9;56:166-173. Epub 2017 Jun 9.

The University of Minnesota, Twin Cities, Department of Genetics, Cell Biology and Development, Masonic Cancer Center, 6-160 Jackson Hall, 321 Church St SE., Minneapolis, MN 55455, United States.

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. With a special focus on mammalian systems, we will then highlight the role of dormant origins in preventing replication-associated genome instability and their functional interplay with proteins involved in the DNA damage repair response for tumor suppression. Lastly, deficiencies in the origin licensing machinery will be discussed in relation to their influence on stem cell maintenance and human diseases.
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http://dx.doi.org/10.1016/j.dnarep.2017.06.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5547906PMC
August 2017

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

Stem Cell Reports 2015 Aug 16;5(2):185-94. Epub 2015 Jul 16.

Yale Stem Cell Center and Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520, USA; SIAIS and School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. Electronic address:

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. However, it is not known whether DOs exist and play important roles in any stem cell type. Here, we show that embryonic stem cells (ESCs) contain more DOs than tissue stem/progenitor cells such as neural stem/progenitor cells (NSPCs). Partial depletion of DOs does not affect ESC self-renewal but impairs their differentiation, including toward the neural lineage. However, reduction of DOs in NSPCs impairs their self-renewal due to accumulation of DNA damage and apoptosis. Furthermore, mice with reduced DOs show abnormal neurogenesis and semi-embryonic lethality. Our results reveal that ESCs are equipped with more DOs to better protect against replicative stress than tissue-specific stem/progenitor cells.
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http://dx.doi.org/10.1016/j.stemcr.2015.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4618655PMC
August 2015

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

Methods Mol Biol 2014 ;1194:341-52

Department of Genetics, Cell Biology and Development, University of Minnesota, 6-160 Jackson Hall, 321 Church St SE, Minneapolis, MN, 55455, USA.

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. Then, we describe the cytokinesis-block micronucleus assay, which can be combined with detection of 53BP1 nuclear bodies to measure the levels of replication-associated genome instability carried over into G1 phase of the cell cycle.
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http://dx.doi.org/10.1007/978-1-4939-1215-5_19DOI Listing
March 2015

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

PLoS Genet 2014 Jul 10;10(7):e1004471. Epub 2014 Jul 10.

Department of Biomedical Sciences, Cornell University, Ithaca, New York, United States of America.

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. The signaling mechanisms underlying hypogonadism and testicular failure in FA patients or mouse models are unknown. We conducted genetic studies to show that hypogonadism of Fancm mutant mice is a result of reduced proliferation, but not apoptosis, of PGCs, resulting in reduced germ cells in neonates of both sexes. Progressive loss of germ cells in adult males also occurs, overlaid with an elevated level of meiotic DNA damage. Genetic studies indicated that ATM-p53-p21 signaling is partially responsible for the germ cell deficiency.
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http://dx.doi.org/10.1371/journal.pgen.1004471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4091704PMC
July 2014

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

Nucleic Acids Res 2014 May 3;42(9):5605-15. Epub 2014 Mar 3.

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA Masonic Cancer Center, Minneapolis, MN 55455, USA

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. To understand this further, we introduced a null allele of Fancc (Fancc-), encoding a member of the FA core complex, into the Mcm4chaos3 background. Primary embryonic fibroblasts double homozygous for Mcm4chaos3 and Fancc- (Mcm4chaos3/chaos3;Fancc-/-) showed significantly increased levels of markers of stalled/collapsed forks compared to either single homozygote. Interestingly, a loss of dormant origins also increased the number of sites in which replication was delayed until prophase, regardless of FA pathway activation. These replication defects coincided with substantially elevated levels of genome instability in Mcm4chaos3/chaos3;Fancc-/- cells, resulting in a high rate of perinatal lethality of Mcm4chaos3/chaos3;Fancc-/- mice and the accelerated tumorigenesis of surviving mice. Together, these findings uncover a specialized role of dormant origins in replication completion while also identifying important functional overlaps between dormant origins and the FA pathway in maintaining fork progression, genome stability, normal development and tumor suppression.
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http://dx.doi.org/10.1093/nar/gku170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027174PMC
May 2014

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

Nucleic Acids Res 2013 Dec 4;41(22):10283-97. Epub 2013 Sep 4.

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA, Molecular, Cellular, Developmental Biology and Genetics Graduate Program, University of Minnesota, Minneapolis, MN 55455, USA, Masonic Cancer Center, Minneapolis, MN 55455, USA and College of Veterinary Medicine, University of Minnesota, St. Paul, MN 55108, USA.

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. Here, we investigated this question in mice using the Helq(gt) and Fancc(-) strains. Compared with Fancc(-)(/)(-) mice lacking FANCC, a component of the FA core complex, Helq(gt/gt) mice exhibited a mild of form of FA-like phenotypes including hypogonadism and cellular sensitivity to the crosslinker mitomycin C. However, unlike Fancc(-)(/)(-) primary fibroblasts, Helq(gt/gt) cells had intact FANCD2 mono-ubiquitination and focus formation. Notably, for all traits examined, Helq was non-epistatic with Fancc, as Helq(gt)(/gt);Fancc(-)(/)(-) double mutants displayed significantly worsened phenotypes than either single mutant. Importantly, this was most noticeable for the suppression of spontaneous chromosome instability such as micronuclei and 53BP1 nuclear bodies, known consequences of persistently stalled replication forks. These findings suggest that mammalian HELQ contributes to genome stability in unchallenged conditions through a mechanism distinct from the function of FANCC.
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http://dx.doi.org/10.1093/nar/gkt676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3905894PMC
December 2013

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

Mamm Genome 2011 Oct 25;22(9-10):506-17. Epub 2011 May 25.

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA.

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. Therefore, it is proposed that the vast majority of MCM2-7 complexes license dormant origins that can be used as backups. Consistent with this idea, it has been repeatedly demonstrated that a reduction (~60%) in chromatin-bound MCM2-7 complexes has little effect on the density of active origins. In this study, however, we describe the first exception to this observation. A reduction of licensed origins due to Mcm4 ( chaos3 ) homozygosity reduces active origin density in primary embryonic fibroblasts (MEFs) in a C57BL/6J (B6) background. We found that this is associated with an intrinsically lower level of active origins in this background compared to others. B6 Mcm4 ( chaos3/chaos3 ) cells proliferate slowly due to p53-dependent upregulation of p21. In fact, the development of B6 Mcm4 ( chaos3/chaos3 ) mice is impaired and a significant fraction of them die at birth. While inactivation of p53 restores proliferation in B6 Mcm4 ( chaos3/chaos3 ) MEFs, it paradoxically does not rescue animal lethality. These findings indicate that a reduction of licensed origins may cause a more profound effect on cell types with lower densities of active origins. Moreover, p53 is required for the development of mice that suffer from intrinsic replication stress.
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http://dx.doi.org/10.1007/s00335-011-9333-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3528403PMC
October 2011

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

Mol Cell 2011 Mar;41(5):543-53

Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455, USA.

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. We found that this allele reduces the stability of the MCM2-7 complex, but confers normal helicase activity in vitro. Despite the activation of multiple fork recovery pathways, replication intermediates in these cells persist into M phase, increasing the number of abnormal anaphase cells with lagging chromosomes and/or acentric fragments. These findings suggest that dormant origins constitute a major pathway for stalled fork recovery, contributing to faithful chromosome segregation and tumor suppression.
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http://dx.doi.org/10.1016/j.molcel.2011.02.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3062258PMC
March 2011

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

Mutat Res 2009 Jun 27;666(1-2):74-8. Epub 2009 Mar 27.

Department of Genetics, Cell Biology and Development, College of Biological Sciences,Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, United States.

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. Previously, we reported the recovery of the first viable Mcm mutant allele (named Mcm4(Chaos3)) in mice. The Mcm4(Chaos3) allele destabilizes the MCM2-7 complex, leading to chromosome instability and the formation of spontaneous tumors in Mcm4(Chaos3) homozygous mice. Supporting our finding, a recent study reported that mice with reduced expression of MCM2 die with lymphomas within the first few months after birth. These data strongly suggest that mutant Mcm2-7 genes are cancer-causing genes with nearly complete penetrance in mice. This could be the case for humans as well. Nevertheless, related investigations have not been undertaken due to the essential nature of the MCM2-7 genes. To circumvent this problem, we focused on the variant alleles of human MCM2-7 genes derived from single nucleotide polymorphisms. We created a total of 14 variant alleles in the corresponding genes in Saccharomyces cerevisiae. The phenotypic consequence was assayed for minichromosome loss, a surrogate phenotype for genome instability and cancer susceptibility. This screen identified a MCM5 variant allele with pathogenic potential. This allele deserves further investigations on its effect on cancer development in human populations.
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http://dx.doi.org/10.1016/j.mrfmmm.2009.03.006DOI Listing
June 2009

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

Cell Cycle 2007 May 5;6(10):1135-40. Epub 2007 May 5.

Department of Genetics, Cell Biology and Development, Medical School and College of Biological Sciences, University of Minnesota, Minneapolis, Minnesota 55455, USA.

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. The Mcm4(Chaos3) mutation appears to destabilize the MCM2-7 complex, causing impaired DNA replication. These findings demonstrate, for the first time, the causative role of an Mcm mutation in cancer development. Furthermore, this raises the possibility that hypomorphic mutations in MCM2-7 genes may increase breast cancer risk in humans.
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http://dx.doi.org/10.4161/cc.6.10.4250DOI Listing
May 2007

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

Nat Genet 2007 Jan 3;39(1):93-8. Epub 2006 Dec 3.

Department of Genetics, Cell Biology and Development, College of Biological Sciences, University of Minnesota, Minneapolis, Minnesota 55455, USA.

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. Saccharomyces cerevisiae strains that we engineered to contain a corresponding allele (resulting in an F391I change) showed a classical minichromosome loss phenotype. Whereas homozygosity for a disrupted Mcm4 allele (Mcm4(-)) caused preimplantation lethality, Mcm(Chaos3/-) embryos died late in gestation, indicating that Mcm4(Chaos3) is hypomorphic. Mutant embryonic fibroblasts were highly susceptible to chromosome breaks induced by the DNA replication inhibitor aphidicolin. Most notably, >80% of Mcm4(Chaos3/Chaos3) females succumbed to mammary adenocarcinomas with a mean latency of 12 months. These findings suggest that hypomorphic alleles of the genes encoding the subunits of the MCM2-7 complex may increase breast cancer risk.
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http://dx.doi.org/10.1038/ng1936DOI Listing
January 2007

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

EMBO J 2005 Nov 13;24(21):3757-69. Epub 2005 Oct 13.

Center for Immunology, School of Medicine and School of Biological Sciences, University of California, Irvine, CA, USA.

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. Here, we show that deletion in C57BL/6J mice of the translesion polymerase theta, which possesses a dual nucleotide mispair inserter-extender function, results in greater than 60% decrease of mutations in antigen-selected V186.2DJ(H) transcripts and greater than 80% decrease in mutations in the Ig H chain intronic J(H)4-iEmu sequence, together with significant alterations in the spectrum of the residual mutations. Thus, polymerase theta plays a dominant role in SHM, possibly by introducing mismatches while bypassing abasic sites generated by UDG-mediated deglycosylation of AID-effected dU, by extending DNA past such abasic sites and by synthesizing DNA during dU:dG mismatch repair.
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http://dx.doi.org/10.1038/sj.emboj.7600833DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1276717PMC
November 2005

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

Mol Cell Biol 2004 Dec;24(23):10381-9

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, 9th Fl. Vet. Research Tower, Ithaca, NY 14853, USA.

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;. We demonstrate here that chaos1 is a mutant allele of Polq by using two genetic approaches: chaos1 mutant phenotype correction by a bacterial artificial chromosome carrying wild-type Polq and a failed complementation test between chaos1 and a Polq-disrupted allele generated by gene targeting. To investigate the potential involvement of Polq in DNA double-strand break repair, we introduced chaos1 into an Atm (for ataxia telangiectasia mutated)-deficient background. The majority ( approximately 90%) of double-homozygous mice died during the neonatal period. Surviving double mutants exhibited synergistic phenotypes such as severe growth retardation and enhanced chromosome instability. However, remarkably, double mutants had delayed onset of thymic lymphoma, significantly increasing life span. These data suggest a unique role of Polq in maintaining genomic integrity, which is probably distinctive from the major homologous recombination pathway regulated by ATM.
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http://dx.doi.org/10.1128/MCB.24.23.10381-10389.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC529050PMC
December 2004

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

Methods Mol Biol 2004 ;262:87-107

The Jackson Laboratory, Bar Harbor, ME, USA.

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. Phenotype-based random mutagenesis of the genome is well suited for this goal. In the mouse, N-ethyl-N-nitrosourea (ENU) induces point mutations at a high frequency in the mouse germline. In this chapter, we describe methods for detecting and characterizing recombination mutations in mice produced by ENU mutagenesis. Potential meiotic recombination mutants are identified in a hierarchical fashion, by performing a screen for infertility, then gonad histology to determine whether meiotic arrest occurs, and finally by immunohistochemical analysis of meiotic chromosome with a battery of antibody markers. Screening for mutations potentially required for recombinational repair of DNA damage in somatic cells is performed using a flow cytometry-based micronucleus assay. Both strategies have proved effective in identifying desired classes of mutations.
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http://dx.doi.org/10.1385/1-59259-761-0:087DOI Listing
March 2004

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

Anat Sci Int 2003 Jun;78(2):85-97

Department of Neurobiology and Anatomy, Kochi Medical School, Nankoku, Kochi, Japan.

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. Detection of ER-beta mRNA using the in situ hybridization method with a digoxigenin-labeled RNA probe was performed in two groups of female rats: ovariectomized (OVX) and estrogen (E2)-treated. A wide distribution of ER-beta mRNA-containing cells was demonstrated in both groups. In the E2-treated group compared with the OVX group, the number of ER-beta mRNA-containing cells was significantly reduced in the external plexiform layer of the olfactory bulb, entorhinal cortex, intermediate part of the lateral septal nucleus, nucleus of the horizontal limb of the diagonal band, amygdala (lateral, medial and basolateral part), thalamus (anteroventral, laterodorsal and lateral posterior part), medial geniculate nucleus, suprachiasmatic nucleus and Purkinje cells in the cerebellum. These results reveal that ER-beta mRNA-containing cells were decreased by estrogen in several brain regions in the female rat brain, suggesting that ER-beta mRNA is downregulated by the physiological level of estrogen in a region-specific manner.
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http://dx.doi.org/10.1046/j.0022-7722.2003.00042.xDOI Listing
June 2003

Phenotype-based identification of mouse chromosome instability mutants.

Genetics 2003 Mar;163(3):1031-40

The Jackson Laboratory, Bar Harbor, Maine 04609, USA.

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. Among offspring derived from males mutagenized with the germline mutagen N-ethyl-N-nitrosourea (ENU), we identified a recessive mutation conferring elevated levels of spontaneous and radiation- or mitomycin C-induced micronuclei. This mutation, named chaos1 (chromosome aberration occurring spontaneously 1), was genetically mapped to a 1.3-Mb interval on chromosome 16 containing Polq, encoding DNA polymerase theta. We identified a nonconservative mutation in the ENU-derived allele, making it a strong candidate for chaos1. POLQ is homologous to Drosophila MUS308, which is essential for normal DNA interstrand crosslink repair and is unique in that it contains both a helicase and a DNA polymerase domain. While cancer susceptibility of chaos1 mutant mice is still under investigation, these data provide a practical paradigm for using a forward genetic approach to discover new potential cancer susceptibility genes using the surrogate biomarker of chromosome instability as a screen.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1462482PMC
March 2003