Distinct parts of minichromosome maintenance protein 2 associate with histone H3/H4 and RNA polymerase II holoenzyme.
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.
Holland
Linda
L
Department of Molecular Biology and Genetics, University of Guelph, Ontario Canada.
Gauthier
Laura
L
Bell-Rogers
Patricia
P
Yankulov
Krassimir
K
D000123:Acetyltransferases / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D002846:Chromatography, Affinity; D019281:Dimerization; D005982:Glutathione Transferase / Q000235:genetics; D051548:Histone Acetyltransferases; D006657:Histones / Q000737:chemistry* / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D064111:Minichromosome Maintenance Complex Component 2; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D012319:RNA Polymerase II / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D017384:Sequence Deletion; D035165:Transcription Factors, General / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D035602:Transcriptional Elongation Factors
D006657:Histones; D020035:Holoenzymes; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D035165:Transcription Factors, General; D035602:Transcriptional Elongation Factors; C018648:transcription factor S-II; D000123:Acetyltransferases; D051548:Histone Acetyltransferases; D005982:Glutathione Transferase; D012319:RNA Polymerase II; D064111:Minichromosome Maintenance Complex Component 2