159 research outputs found
Gated rotation mechanism of site-specific recombination by ĻC31 integrase
Integrases, such as that of the Streptomyces temperate bacteriophage ĻC31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ĻC31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a āsubunit rotationā mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining att sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ĻC31 integrase-mediated recombination in a topologically constrained experimental system using hybrid āphesā recombination sites, each of which comprises a ĻC31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180Ā° rotation. However, multiple processive ā360Ā° rotationā rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory āgatingā mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round
Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Endoreplication Cycles
Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. We examined the behavior of MCM proteins in endoreplicating larval salivary glands, to determine whether oscillation of MCMāchromosome localization occurs in conjunction with passage through an endocycle S phase. We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes. We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association. We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCMāchromosome association normally occur in endocycles. These results are discussed in a model in which the cycle of MCMāchromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles
Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Mitotic Cycles
Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Their absence on chromatin is thought to contribute to the inability of a G2 nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCMāchromatin interaction, we tested whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B showed that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. dacapo mutant embryos lack an inhibitor of cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. We propose that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. We suggest that cyclins have both positive and negative roles in controlling MCMāchromatin association
The Hin recombinase assembles a tetrameric protein swivel that exchanges DNA strands
Most site-specific recombinases can be grouped into two structurally and mechanistically different classes. Whereas recombination by tyrosine recombinases proceeds with little movements by the proteins, serine recombinases exchange DNA strands by a mechanism requiring large quaternary rearrangements. Here we use site-directed crosslinking to investigate the conformational changes that accompany the formation of the synaptic complex and the exchange of DNA strands by the Hin serine recombinase. Efficient crosslinking between residues corresponding to the āD-helixā region provides the first experimental evidence for interactions between synapsed subunits within this region and distinguishes between different tetrameric conformers that have been observed in crystal structures of related serine recombinases. Crosslinking profiles between cysteines introduced over the 35 residue E-helix region that constitutes most of the proposed rotating interface both support the long helical structure of the region and provide strong experimental support for a subunit rotation mechanism that mediates DNA exchange
Performance of the colorectal cancer screening marker Sept9 is influenced by age, diabetes and arthritis: a nested caseācontrol study
Molecular analysis of selected cell cycle regulatory proteins during aerobic and hypoxic maintenance of human ovarian carcinoma cells
Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Endoreplication Cycles
Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Mitotic Cycles
Exploring DNA methylation changes in promoter, intragenic, and intergenic regions as early and late events in breast cancer formation
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