Germline mutations in the proof-reading domains of POLE and POLD1 predispose to colorectal adenomas and carcinomas.

Many individuals with multiple or large colorectal adenomas or early-onset colorectal cancer (CRC) have no detectable germline mutations in the known cancer predisposition genes. Using whole-genome sequencing, supplemented by linkage and association analysis, we identified specific heterozygous POLE or POLD1 germline variants in several multiple-adenoma and/or CRC cases but in no controls. The variants associated with susceptibility, POLE p.Leu424Val and POLD1 p.Ser478Asn, have high penetrance, and POLD1 mutation was also associated with endometrial cancer predisposition. The mutations map to equivalent sites in the proofreading (exonuclease) domain of DNA polymerases ɛ and δ and are predicted to cause a defect in the correction of mispaired bases inserted during DNA replication. In agreement with this prediction, the tumors from mutation carriers were microsatellite stable but tended to acquire base substitution mutations, as confirmed by yeast functional assays. Further analysis of published data showed that the recently described group of hypermutant, microsatellite-stable CRCs is likely to be caused by somatic POLE mutations affecting the exonuclease domain.post-print535 K

Differences in the degree of inhibition of NDP reductase by chemical inactivation and by the thermosensitive mutation nrdA101 in Escherichia coli suggest an effect on chromosome segregation.

NDP reductase activity can be inhibited either by treatment with hydroxyurea or by incubation of an nrdAts mutant strain at the non-permissive temperature. Both methods inhibit replication, but experiments on these two types of inhibition yielded very different results. The chemical treatment immediately inhibited DNA synthesis but did not affect the cell and nucleoid appearance, while the incubation of an nrdA101 mutant strain at the nonpermissive temperature inhibited DNA synthesis after more than 50 min, and resulted in aberrant chromosome segregation, long filaments, and a high frequency of anucleate cells. These phenotypes are not induced by SOS. In view of these results, we suggest there is an indirect relationship between NDP reductase and the chromosome segregation machinery through the maintenance of the proposed replication hyperstructure.post-print734 K

Relationship between Fork Progression and Initiation of Chromosome Replication in E. coli

post-print687 K

Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2.

The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the 'Y pocket') that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases.We are grateful for the access and support at beamlines i02, i03 and i04-1 at Diamond Light Source at Harwell, UK (proposal MX9007 and MX9537) and at beamline Proxima1 at the SOLEIL Synchrotron, Gif-sur-Yvette, France. We are grateful for access and support from the X-ray and biophysics facilities (Dept. of Biochemistry) and the screening/imaging facility (MRC Cancer Unit). M.J. was supported by a Cancer Research UK studentship held in the labs of DS and ARV, PS and MR by a Wellcome Trust Strategic Award to ARV and MH, and DJH, BH, AJN and GM by grants from the UK Medical Research Council to ARV.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/srep2852

Implicaciones de la NDP reductasa en la progresión de las horquillas de replicación en Escherichia coli

NDP reductase is the only specific enzyme required for the enzymatic synthesis of the precursors of DNA and it is a part of the Nucleotides Synthesis Complex. In this work we have analysed the replication fork progression in a nrdA101 mutant, which has a thermosensitive NDP reductase. We have proved that the Nucleotides Synthesis Complex and the Replication Complex are associated in a Replication Hyperstructure.La NDP reductasa es la única enzima requerida directamente para la biosíntesis de los precursores del DNA y está formando parte del Complejo de Síntesis de Nucleótidos. En este trabajo se ha analizado la progresión de las horquillas de replicación en un mutante nrdA101 que posee un NDP reductasa termosensible, comprobándose que el Complejo se Síntesis de Nucleótidos y el de Replicación están asociados formando una Hiperestructura de Replicación

A kinase-independent function for AURORA-A in replisome assembly during DNA replication initiation.

The catalytic activity of human AURORA-A kinase (AURKA) regulates mitotic progression, and its frequent overexpression in major forms of epithelial cancer is associated with aneuploidy and carcinogenesis. Here, we report an unexpected, kinase-independent function for AURKA in DNA replication initiation whose inhibition through a class of allosteric inhibitors opens avenues for cancer therapy. We show that genetic depletion of AURKA, or its inhibition by allosteric but not catalytic inhibitors, blocks the G1-S cell cycle transition. A catalytically inactive AURKA mutant suffices to overcome this block. We identify a multiprotein complex between AURKA and the replisome components MCM7, WDHD1 and POLD1 formed during G1, and demonstrate that allosteric but not catalytic inhibitors prevent the chromatin assembly of functional replisomes. Indeed, allosteric but not catalytic AURKA inhibitors sensitize cancer cells to inhibition of the CDC7 kinase subunit of the replication-initiating factor DDK. Thus, our findings define a mechanism essential for replisome assembly during DNA replication initiation that is vulnerable to inhibition as combination therapy in cancer

Cellular regulation of ribonucleotide reductase in eukaryotes.

Synthesis of deoxynucleoside triphosphates (dNTPs) is essential for both DNA replication and repair and a key step in this process is catalyzed by ribonucleotide reductases (RNRs), which reduce ribonucleotides (rNDPs) to their deoxy forms. Tight regulation of RNR is crucial for maintaining the correct levels of all four dNTPs, which is important for minimizing the mutation rate and avoiding genome instability. Although allosteric control of RNR was the first discovered mechanism involved in regulation of the enzyme, other controls have emerged in recent years. These include regulation of expression of RNR genes, proteolysis of RNR subunits, control of the cellular localization of the small RNR subunit, and regulation of RNR activity by small protein inhibitors. This review will focus on these additional mechanisms of control responsible for providing a balanced supply of dNTPs.pre-print759 K

Development of a new genotyping assay for detection of the BDNF Val66Met polymorphism using melting-curve analysis.

Brain-derived neurotrophic factor (BDNF) plays a critical role in the growth, differentiation and survival of neurons in the CNS. Recent research has suggested that BDNF may be implicated in the etiology of mood disorders and schizophrenia, as well as in the therapeutic action of some drugs, such as antidepressants and antipsychotics. This study aimed to develop a simple, fast and accurate new method for detecting the Val66Met polymorphism of the BDNF gene in schizophrenia patients using melting-curve analysis and a DNA-specific dye, SYBR® Green I. A group of 30 schizophrenia patients were analyzed to detect the BDNF Val66Met polymorphism (rs6265) using the new genotyping method based on the analysis of fluorescence melting curves of PCR products that were labeled with SYBR Green I. The genotype results were confirmed for all 30 samples using the specific BDNF TaqMan® allele discrimination assay. This new method allows the analysis of both alleles in the same reaction tube using SYBR Green I, with no need for additional steps. The addition of a GC clamp makes this method universally applicable, since the melting temperature of one allele can be adjusted as necessary to give the distinctive separation of melting curves. Therefore, this new method is simple, fast and accurate for determining the presence of the BDNF Val66Met polymorphism. It may also be useful for the analysis of other SNPs in pharmacogenetic studiespost-print1390 K

Processing of stalled replication forks under thymine starvation and its relationship with thymine less death in Escherichia coli.

pre-prin

Riboucleoside diphosphate reductase is a functional and structural component of the replication hyperstructure in Escherichia coli.

pre-prin