Distinct parts of minichromosome maintenance protein 2 associate with histone H3/H4 and RNA polymerase II holoenzyme

Abstract

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][3][4][5][6][7][8]. They are referred to as pol II ÔholoenzymeÕ. 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 against the CTD disrupt the yeast holoenzyme into core pol II and a mediator subcomplex, which contains the SRB and MED proteins [7][8][9]. A similar treatment of pol II holoenzyme from HeLa cells also disrupts its interaction with several of the GTFs [6]. In higher eukaryotes the CTD mediates the interaction with complexes that contain homologues of the yeast SRB and MED proteins such as SMCC [10] or NAT [11]. It is believed that pol II holoenzyme is a functionally significant complex, which is responsible for transactivator-stimulated transcription in vivo. It has been shown that the srb4 and srb6 genes are essential for expression of most mRNAs in budding yeast [12]. Other holoenzyme components such as SRB 2, 5, 7-11, SWI/SNF proteins, SIN4, RGR1, MED2, MED9/CSE2, MED10/NUT2, MED11, GAL11, PGD1 and ROX3 [7,9,[13][14][15][16] are not essential for transcription of most genes but do contribute to the response to transactivators and repressors (reviewed in [17,18]). In addition to its role in the response to transcriptional regulators, pol II holoenzyme may be involved in integrating transcription with RNA processing, DNA repair and replication. In support of this idea, the DNA repair factors DNA pol e, XPC, XPF, XPG, Ku, RAD51 [3], BRCA1 [19]; RNA helicase A [20]; the replication factors RP-A, RP-C [3] and MCM proteins [6]; and the cleavage/polyadenylation factors CPSF and CstF [21] have been id...

The DNA replication factor MCM5 is essential for Stat1-mediated transcriptional activation

“…Our results would suggest that the MCM2-MCM7 proteins travel along with the RNA Pol II to unwind DNA during transcription elongation just as they do for DNA replication elongation (4,5). The interaction between MCM2 and the Pol II C-terminal domain (26,27) would provide an anchor and guide for the MCM2-MCM7 hexamer to move along the DNA template during transcription elongation.…”

“…Gel filtration analyses showed that the MCM3͞5 subcomplex coeluted with the active tyrosine-phosphorylated Stat1 when cells were stimulated with IFN-␥ (25). Other interactions between members of the MCM family and proteins involved in transcription activation were also identified, including MCM2 with the RNA polymerase II (26,27) and MCM7 with Rb (28). However, it is not clear whether these interactions result in the participation of MCM proteins in transcription or these transcription factors may modulate DNA replication.…”

MCM proteins and checkpoint kinases get together at the fork

“…However, an argument against a direct inhibitory role of MCM2 phosphorylation by ATR on helicase function is the fact that aphidicolin treatment, which uncouples DNA polymerases from the helicase, leads to the generation of extensive regions of ssDNA (15), suggesting that the replicative helicase is not directly inhibited in these conditions. Additionally, phosphorylation of MCM2 in the N-terminal region could affect its known interactions with the transcription machinery (16,17). MCM3 is phosphorylated by ATM at S535 located between the modules corresponding to box VII and VIIЈ of AAA ϩ protein (4).…”

The Coordination between DNA Replication Initiation and Other Cell Cycle Events

“…The association of MCMs with the holoenzyme partly depends on its amino acids 168-230 and the C-terminal domain of Pol II (Yankulov et al 1999). Mutations in amino acids 169-212 of MCM2 disrupt its binding to Pol II and to general transcription factors in vivo (Holland et al 2002). MCM2 and MCM5 are also required for general transcription, and their depletion may lead to transcription defects.…”