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      Polε Instability Drives Replication Stress, Abnormal Development, and Tumorigenesis


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          DNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains. Comparable phenotypes of growth retardation and immunodeficiency are also observed in human patients harboring destabilizing mutations in POLE1. In both Pole4 −/− mouse and POLE1 mutant human cells, Polε hypomorphy is associated with replication stress and p53 activation, which we attribute to inefficient replication origin firing. Strikingly, removing p53 is sufficient to rescue embryonic lethality and all developmental abnormalities in Pole4 null mice. However, Pole4 −/− p53 +/− mice exhibit accelerated tumorigenesis, revealing an important role for controlled CMG and origin activation in normal development and tumor prevention.

          Graphical Abstract


          • Pole4 −/− mice exhibit impaired development and defective lymphocyte maturation

          • Pole4 is crucial for maintaining the stability of the Polε complex

          • Polε hypomorphy causes replication stress via inefficient origin activation

          • p53 inactivation rescues all major developmental defects in Pole4-deficient mice


          Bellelli et al. report that Pole4 deficiency in mice or POLE1 mutations in humans result in Pol Epsilon complex instability, replication stress, and inefficient origin activation. As a consequence, Pol Epsilon hypomorphic mice exhibit aberrant growth and development, lymphopenia, and tumor predisposition, which can be rescued by deleting p53.

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          Most cited references 48

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          Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours.

          Mutations in the p53 tumour-suppressor gene are the most frequently observed genetic lesions in human cancers. To investigate the role of the p53 gene in mammalian development and tumorigenesis, a null mutation was introduced into the gene by homologous recombination in murine embryonic stem cells. Mice homozygous for the null allele appear normal but are prone to the spontaneous development of a variety of neoplasms by 6 months of age. These observations indicate that a normal p53 gene is dispensable for embryonic development, that its absence predisposes the animal to neoplastic disease, and that an oncogenic mutant form of p53 is not obligatory for the genesis of many types of tumours.
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            Tumor spectrum analysis in p53-mutant mice.

            The p53 tumor suppressor gene is mutated in a large percentage of human malignancies, including tumors of the colon, breast, lung and brain. Individuals who inherit one mutant allele of p53 are susceptible to a wide range of tumor types. The gene encodes a transcriptional regulator that may function in the cellular response to DNA damage. The construction of mouse strains carrying germline mutations of p53 facilitates analysis of the function of p53 in normal cells and tumorigenesis. In order to study the effects of p53 mutation in vivo, we have constructed a mouse strain carrying a germline disruption of the gene. This mutation removes approximately 40% of the coding capacity of p53 and completely eliminates synthesis of p53 protein. As observed previously for a different germline mutation of p53, animals homozygous for this p53 deletion mutation are viable but highly predisposed to malignancy. Heterozygous animals also have an increased cancer risk, although the distribution of tumor types in these animals differs from that in homozygous mutants. In most cases, tumorigenesis in heterozygous animals is accompanied by loss of the wild-type p53 allele. We reaffirm that p53 function is not required for normal mouse development and conclude that p53 status can strongly influence tumor latency and tissue distribution.
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              Regulated Eukaryotic DNA Replication Origin Firing with Purified Proteins

              Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric MCM complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45, MCM, GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4 dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

                Author and article information

                Mol Cell
                Mol. Cell
                Molecular Cell
                Cell Press
                17 May 2018
                17 May 2018
                : 70
                : 4
                : 707-721.e7
                [1 ]The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
                [2 ]MRC Institute of Genetics & Molecular Medicine, The University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
                [3 ]Department of Genetic Medicine, St Mary’s Hospital, Oxford Road, Manchester, M13 OJH, UK
                [4 ]Department of Paediatrics, Cork University Hospital, Wilton, Cork T12 DC4A, Ireland
                Author notes
                []Corresponding author simon.boulton@ 123456crick.ac.uk

                These authors contributed equally


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                © 2018 The Author(s)

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).



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