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      Functional Conservation of the Pre-Sensor One Beta-Finger Hairpin (PS1-hp) Structures in Mini-Chromosome Maintenance Proteins of Saccharomyces cerevisiae and Archaea

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          Abstract

          Mini-chromosome maintenance (MCM) proteins form complexes that are required for DNA replication and are highly conserved throughout evolution. The replicative helicase of eukaryotic organisms is composed of the six paralogs MCM2-7, which form a heterohexameric ring structure. In contrast, the structure of the archaean replicative MCM helicase is a single Mcm protein that forms a homohexameric complex. Atomic structures of archaeal MCMs have identified multiple beta-finger structures in Mcm proteins whose in vivo function is unknown. In the present study, we have investigated the physiological role of the pre-sensor 1 beta -hairpin (PS1-hp) beta-fingers of Saccharomyces cerevisiae Mcm4p and Mcm5p in DNA replication initiation and elongation in vivo. The PS1-hp beta-finger mutant of Mcm5p ( mcm5-HAT K506A :: URA3 ) has a growth defect at both 18° and 37°. Mutation of the Mcm4p PS1-hp beta-finger ( mcm4-HA K658A :: TRP1 ) does not have a growth defect, indicating different functional contributions of the PS1-hp beta-finger structures of different MCM helicase subunits. Both Mcm4p and Mcm5p PS1-hp beta-finger mutants can coimmunoprecipitate Mcm2p, indicating the formation of the hexameric MCM helicase complex. Both PS1-hp beta-finger mutants have a plasmid loss phenotype that is suppressible by origin dosage, indicating a defective replication initiation. Surprisingly, a defect in the binding of PS1-hp MCM mutants to origins of DNA replication was not found by chromatin immunoprecipitation, suggesting a novel interpretation in which the defect is in a subsequent step of DNA strand separation by the MCM helicase. The double mutant mcm4-HA K658A :: TRP1 mcm5-HAT K506A :: URA3 is lethal, displaying a terminal MCM mutant phenotype of large budded cells.

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          Most cited references 37

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          DNA replication in eukaryotic cells.

          The maintenance of the eukaryotic genome requires precisely coordinated replication of the entire genome each time a cell divides. To achieve this coordination, eukaryotic cells use an ordered series of steps to form several key protein assemblies at origins of replication. Recent studies have identified many of the protein components of these complexes and the time during the cell cycle they assemble at the origin. Interestingly, despite distinct differences in origin structure, the identity and order of assembly of eukaryotic replication factors is highly conserved across all species. This review describes our current understanding of these events and how they are coordinated with cell cycle progression. We focus on bringing together the results from different organisms to provide a coherent model of the events of initiation. We emphasize recent progress in determining the function of the different replication factors once they have been assembled at the origin.
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            Replication dynamics of the yeast genome.

            Oligonucleotide microarrays were used to map the detailed topography of chromosome replication in the budding yeast Saccharomyces cerevisiae. The times of replication of thousands of sites across the genome were determined by hybridizing replicated and unreplicated DNAs, isolated at different times in S phase, to the microarrays. Origin activations take place continuously throughout S phase but with most firings near mid-S phase. Rates of replication fork movement vary greatly from region to region in the genome. The two ends of each of the 16 chromosomes are highly correlated in their times of replication. This microarray approach is readily applicable to other organisms, including humans.
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              Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing.

              The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs). Mcm2-7 forms the core of the replicative DNA helicase, which is inactive in the pre-RC. The loading of Mcm2-7 onto DNA requires the origin recognition complex (ORC), Cdc6, and Cdt1, and depends on ATP. We have reconstituted Mcm2-7 loading with purified budding yeast proteins. Using biochemical approaches and electron microscopy, we show that single heptamers of Cdt1*Mcm2-7 are loaded cooperatively and result in association of stable, head-to-head Mcm2-7 double hexamers connected via their N-terminal rings. DNA runs through a central channel in the double hexamer, and, once loaded, Mcm2-7 can slide passively along double-stranded DNA. Our work has significant implications for understanding how eukaryotic DNA replication origins are chosen and licensed, how replisomes assemble during initiation, and how unwinding occurs during DNA replication.
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                Author and article information

                Journal
                G3 (Bethesda)
                Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes, Genomes, Genetics
                G3: Genes|Genomes|Genetics
                Genetics Society of America
                2160-1836
                23 May 2014
                July 2014
                : 4
                : 7
                : 1319-1326
                Affiliations
                Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045
                Author notes

                Supporting information is available online at http://www.g3journal.org/lookup/suppl/doi:10.1534/g3.114.011668/-/DC1

                [1 ]Corresponding author: 12801 E.17 th Ave, MS8101, University of Colorado, Aurora, CO 80045. E-mail: Robert.sclafani@ 123456ucdenver.edu
                Article
                GGG_011668
                10.1534/g3.114.011668
                4455780
                24875627
                Copyright © 2014 Ramey and Sclafani

                This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License ( http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Page count
                Pages: 8
                Product
                Categories
                Investigations
                Custom metadata
                v1

                Genetics

                replication, yeast, cell cycle, s phase, helicase

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