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      Dual targeting of Saccharomyces cerevisiae Pso2 to mitochondria and the nucleus, and its functional relevance in the repair of DNA interstrand crosslinks

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          Abstract

          Repair of DNA interstrand crosslinks involves a functional interplay among different DNA surveillance and repair pathways. Previous work has shown that interstrand crosslink-inducing agents cause damage to Saccharomyces cerevisiae nuclear and mitochondrial DNA, and its pso2/ snm1 mutants exhibit a petite phenotype followed by loss of mitochondrial DNA integrity and copy number. Complex as it is, the cause and underlying molecular mechanisms remains elusive. Here, by combining a wide range of approaches with in vitro and in vivo analyses, we interrogated the subcellular localization and function of Pso2. We found evidence that the nuclear-encoded Pso2 contains 1 mitochondrial targeting sequence and 2 nuclear localization signals (NLS1 and NLS2), although NLS1 resides within the mitochondrial targeting sequence. Further analysis revealed that Pso2 is a dual-localized interstrand crosslink repair protein; it can be imported into both nucleus and mitochondria and that genotoxic agents enhance its abundance in the latter. While mitochondrial targeting sequence is essential for mitochondrial Pso2 import, either NLS1 or NLS2 is sufficient for its nuclear import; this implies that the 2 nuclear localization signal motifs are functionally redundant. Ablation of mitochondrial targeting sequence abrogated mitochondrial Pso2 import, and concomitantly, raised its levels in the nucleus. Strikingly, mutational disruption of both nuclear localization signal motifs blocked the nuclear Pso2 import; at the same time, they enhanced its translocation into the mitochondria, consistent with the notion that the relationship between mitochondrial targeting sequence and nuclear localization signal motifs is competitive. However, the nuclease activity of import-deficient species of Pso2 was not impaired. The potential relevance of dual targeting of Pso2 into 2 DNA-bearing organelles is discussed.

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          A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

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            Molecular Cloning : A Laboratory Manual

            <p>The first two editions of this manual have been mainstays of molecular biology for nearly twenty years, with an unrivalled reputation for reliability, accuracy, and clarity.<br>In this new edition, authors Joseph Sambrook and David Russell have completely updated the book, revising every protocol and adding a mass of new material, to broaden its scope and maintain its unbeatable value for studies in genetics, molecular cell biology, developmental biology, microbiology, neuroscience, and immunology.<br>Handsomely redesigned and presented in new bindings of proven durability, this three–volume work is essential for everyone using today’s biomolecular techniques.<br>The opening chapters describe essential techniques, some well–established, some new, that are used every day in the best laboratories for isolating, analyzing and cloning DNA molecules, both large and small.<br>These are followed by chapters on cDNA cloning and exon trapping, amplification of DNA, generation and use of nucleic acid probes, mutagenesis, and DNA sequencing.<br>The concluding chapters deal with methods to screen expression libraries, express cloned genes in both prokaryotes and eukaryotic cells, analyze transcripts and proteins, and detect protein–protein interactions.<br>The Appendix is a compendium of reagents, vectors, media, technical suppliers, kits, electronic resources and other essential information.<br>As in earlier editions, this is the only manual that explains how to achieve success in cloning and provides a wealth of information about why techniques work, how they were first developed, and how they have evolved. </p>
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              Mechanisms of DNA damage, repair, and mutagenesis.

              Living organisms are continuously exposed to a myriad of DNA damaging agents that can impact health and modulate disease-states. However, robust DNA repair and damage-bypass mechanisms faithfully protect the DNA by either removing or tolerating the damage to ensure an overall survival. Deviations in this fine-tuning are known to destabilize cellular metabolic homeostasis, as exemplified in diverse cancers where disruption or deregulation of DNA repair pathways results in genome instability. Because routinely used biological, physical and chemical agents impact human health, testing their genotoxicity and regulating their use have become important. In this introductory review, we will delineate mechanisms of DNA damage and the counteracting repair/tolerance pathways to provide insights into the molecular basis of genotoxicity in cells that lays the foundation for subsequent articles in this issue. Environ. Mol. Mutagen., 2017. © 2017 Wiley Periodicals, Inc.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                G3 (Bethesda)
                Genetics
                g3journal
                G3: Genes|Genomes|Genetics
                Oxford University Press
                2160-1836
                June 2022
                28 April 2022
                28 April 2022
                : 12
                : 6
                : jkac066
                Affiliations
                Department of Biochemistry, Indian Institute of Science , Bangalore 560 012, India
                Author notes
                Corresponding author: Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India. Email: kmbc@ 123456iisc.ac.in
                Author information
                https://orcid.org/0000-0003-1381-0363
                https://orcid.org/0000-0002-9192-9194
                Article
                jkac066
                10.1093/g3journal/jkac066
                9157068
                35482533
                80edafdf-9a37-47db-a6f3-7d868c30db56
                © The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 15 March 2022
                : 10 January 2022
                : 05 May 2022
                Page count
                Pages: 18
                Categories
                Investigation
                AcademicSubjects/SCI01180
                AcademicSubjects/SCI01140
                AcademicSubjects/SCI00010
                AcademicSubjects/SCI00960

                Genetics
                pso2,icl repair,genotoxic stress,nucleo-mitochondrial localization,functional redundancy,nuclease activity

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