We report on the characterization of the DNA primase complex of the hyperthermophilic archaeon Pyrococcus abyssi (Pab). The Pab DNA primase complex is composed of the proteins Pabp41 and Pabp46, which show sequence similarities to the p49 and p58 subunits, respectively, of the eukaryotic polymerase alpha-primase complex. Both subunits were expressed, purified, and characterized. The Pabp41 subunit alone had no RNA synthesis activity but could synthesize long (up to 3 kb) DNA strands. Addition of the Pabp46 subunit increased the rate of DNA synthesis but decreased the length of the DNA fragments synthesized and conferred RNA synthesis capability. Moreover, in our experimental conditions, Pab DNA primase had comparable affinities for ribonucleotides and deoxyribonucleotides, and its activity was dependent on the presence of Mg2+ and Mn2+. Interestingly, Pab DNA primase also displayed DNA polymerase, gap-filling, and strand-displacement activities. Genetic analyses undertaken in Haloferax volcanii suggested that the eukaryotic-type heterodimeric primase is essential for survival in archaeal cells. Our results are in favor of a multifunctional archaeal primase involved in priming and repair.

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Eukaryotic elongation factor 1 (eEF1) is a translational multimolecular complex reported in higher eukaryotes to be a target of CDK1/cyclin B, the universal regulator of M phase, but whose role in the cell cycle remains to be determined. A specific polyclonal antibody was produced and used to characterize the delta subunit of sea urchin elongation factor 1 (SgEF1delta) in early embryos, a powerful model for investigating cell cycle regulation. The SgEF1delta protein was present in unfertilized eggs as two isoforms of 35 and 37 kDa, issued from two different mRNAs. The two canonical eEF1delta partners, eEF1gamma and eEF1beta, were shown to co-immunoprecipitate with the SgEF1delta isoforms. Both isoforms were associated in a macromolecular complex, which resolved upon gel filtration chromatography at a molecular weight > 400 kDa, suggesting association with other yet unidentified partners. After fertilization, the amount as well as the ratio of both SgEF1delta isoforms remained constant during the first cell division as judged by Western blotting. Immunofluorescence analysis showed that a pool of the protein concentrated as a ring at the embryo nuclear location around the period of nuclear envelope breakdown and was visualized later as two large spheres around the mitotic spindle poles. Thus, the eEF1delta protein shows cell cycle-specific localization changes in sea urchin embryos.

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The expression and activity of DNA-dependent protein kinase (DNA-PK) is related to DNA repair status in the response of cells to exogenous and endogenous factors. Recent studies indicate that Epidermal Growth Factor Receptor (EGFR) is involved in modulating DNA-PK. It has been shown that a compound 4-nitro-7-[(1-oxidopyridin-2-yl)sulfanyl]-2,1,3-benzoxadiazole (NSC), bearing a nitro-benzoxadiazole (NBD) scaffold, enhances tyrosine phosphorylation of EGFR and triggers downstream signaling pathways. Here, we studied the behavior of DNA-PK and other DNA repair proteins in prostate cancer cells exposed to compound NSC. We showed that both the expression and activity of DNA-PKcs (catalytic subunit of DNA-PK) rapidly decreased upon exposure of cells to the compound. The decline in DNA-PKcs was associated with enhanced protein ubiquitination, indicating the activation of cellular proteasome. However, pretreatment of cells with thioglycerol abolished the action of compound NSC and restored the level of DNA-PKcs. Moreover, the decreased level of DNA-PKcs was associated with the production of intracellular hydrogen peroxide by stable dimeric forms of Cu/Zn SOD1 induced by NSC. Our findings indicate that reactive oxygen species and electrophilic intermediates, generated and accumulated during the redox transformation of NBD compounds, are primarily responsible for the rapid modulation of DNA-PKcs functions in cancer cells.

Additional information is available at the end of the chapter http://dx.doi.org/10.5772/53973 . IntroductionCellular DNA is constantly exposed to the effects of endogenous or environmental agents such as free radicals, radiation and chemicals. In higher organisms, these nucleic alterations are estimated at several thousands of lesions per cell [ ] which can correspond to the loss of bases and also to the breaking of one or both strands of the DNA double helix. Among these DNA breaks, the double-strand break DSB is the most harmful because it is the most difficult to repair. A human cell can accumulate up to DSBs per cell cycle [ ]. Unrepaired DSBs can have serious consequences such as permanent cell cycle arrest or cell death by apoptosis. Imperfect repair can also lead to major syndromes such as genetic disorders, premature aging or malignant cell generation.In response to DNA damage, the cell has developed a surveillance and DNA repair network. DSBs of DNA, which are the most severe nucleic acid alterations, are repaired mainly by either non-homologous end-joining NHEJ or homologous recombination HR .NHEJ repair leads to a direct rejoining of the separated DNA ends [ ]. This pathway begins by the binding of the Ku / heterodimer to DNA ends Figure ] which recruits and induces the activation of the DNA-dependent protein kinase catalytic subunit DNA-PKc . Kinase activity is required for NHEJ since it causes the recruitment of other proteins and promotes the bringing together of DNA ends. Finally, ligase VI and XRCC /XLF co-factors are involved in the final step of ligation and the generation of DNA repair. This process involves mainly the G -G and S phases of the cell cycle. Its disadvantage is the possible loss of genetic information due to deletions or insertions of nucleic acids during the ligation of DNA ends and thus NHEJ repair is considered error-prone. DNA repair by HR is more complex and needs a homologous sequence, which can be present in the homologous chromosome or in a gene in multicopy [ ]. HR predominates in the S and G phases, when the sister chromatids are present and can also be a model for DNA repair [ ]. In eukaryotic cells, DNA repair is supported by several protein complexes. Protein ATM Ataxia Telangiectasia Mutated has a role in DSB signaling via its activation induced by the MRN protein complex MRE -Rad -NBS complex . MRE is a '-' exonuclease that leads to a ' end of DNA which is required for the process [ ]. This resection of single-stranded DNA is followed by the recruitment of many proteins such as RPA, BRCA , BRCA , Rad , Rad , and Rad . Rad is one of the first to settle on the DSB. BRCA then recruits BRCA , Rad and Rad to form the nucleoprotein filament with ssDNA, whose role is to move the blade to the homologous sequence required for HR. Rad protein is the main element involved in the HR process. This recombinase catalyzes the homology search and the strand exchange with a homologous sequence and thus ensures the accurate repair of the DSB. In eukaryotes, Rad recombinase RecA homol...

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