TAILIEUCHUNG - Báo cáo Y học: Distinct parts of minichromosome maintenance protein 2 associate with histone H3/H4 and RNA polymerase II holoenzyme

Minichromosomemaintenance (MCM) proteins are part of the replication licensing factor (RLF-M), which limits the initiationofDNAreplication to once per cell have previously reported that higher order complexes of mam-malian pol II and general pol II transcription factors, referred to as pol II holoenzyme, also contain MCM pro-teins. In the present study we have analyzed in detail the interaction between MCM2 and pol II holoenzyme. N- and C- terminal deletions were introduced into epitope-tagged MCM2 and the truncated proteins were transiently expressed in 293 cells. . | Eur. J. Biochem. 269 5192-5202 2002 FEBS 2002 doi Distinct parts of minichromosome maintenance protein 2 associate with histone H3 H4 and RNA polymerase II holoenzyme Linda Holland Michael Downey Xiaomin Song Laura Gauthier Patricia Bell-Rogers and Krassimir Yankulov Department of Molecular Biology and Genetics University of Guelph Ontario Canada Minichromosome maintenance MCM proteins are part of the replication licensing factor RLF-M which limits the initiation of DNA replication to once per cell cycle. We have previously reported that higher order complexes of mammalian pol II and general pol II transcription factors referred to as pol II holoenzyme also contain MCM proteins. In the present study we have analyzed in detail the interaction between MCM2 and pol II holoenzyme. N- and C- terminal deletions were introduced into epitope-tagged MCM2 and the truncated proteins were transiently expressed in 293 cells. Affinity chromatography was used to purify RNA pol II holoenzyme and histone binding MCM complexes. We found that amino acids 168-230 of MCM2 are required for its binding to pol II holoenzyme in vivo. We also showed that bacterially expressed amino acids 169-212 of MCM2 associate with pol II and several general transcription factors in vitro. Point mutations within the 169-212 domain of MCM2 disrupted its interaction with pol II holoenzyme both in vitro and in vivo. This region is distinct from the previously characterized histone H3 binding domain of MCM2. Keywords MCM2 RNA polymerase II holoenzyme histone. Large protein complexes which contain RNA polymerase II as well as the general pol II transcription factors GTFs TFII A B D E F and H 1 and other proteins have been isolated from yeast mammalian and amphibian cells 2-8 . They are referred to as pol II holoenzyme . Some of the components of pol II holoenzyme make direct or indirect contacts with the C-terminal domain CTD of the largest subunit of pol II. Antibodies .

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