Constitutive association of Mcm2-3-5 proteins with chromatin in Entamoeba histolytica

Eukaryotic cells duplicate their genome once and only once per cell cycle. Our earlier studies with the protozoan parasite, Entamoeba histolytica, have shown that genome reduplication may occur several times without nuclear or cellular division. The Mcm2-7 protein complex is required for licensing of DNA replication. In an effort to understand whether genome reduplication occurs due to absence or failure of the DNA replication licensing system, we analysed the function of Mcm2-3-5 proteins in E. histolytica. In this study, we have cloned E. histolytica (Eh) MCM2 and Eh MCM5 genes, while Eh MCM3 was cloned earlier. The sequence of Eh MCM2-3-5 genes is well conserved with other eukaryotic homologues. We have shown that Eh Mcm2,3 proteins are functional in Saccharomyces cerevisiae. Our studies in E. histolytica showed that Eh Mcm2-3-5 proteins are associated with chromatin constitutively in cycling cells and during arrest of DNA synthesis induced by serum starvation. Alternation of genome duplication with mitosis is regulated by association-dissociation of Mcm2-7 proteins with chromatin in other eukaryotes. Our results suggest that constitutive association of Mcm proteins with chromatin could be one of the reasons why genome reduplication occurs in E. histolytica.     

The genomic instability of yeast cdc6-1/cdc6-1 mutants involves chromosome structure and recombination

When diploid cells of Saccharomyces cerevisiae homozygous for the temperature-sensitive cell division cycle mutation cdc6-1 are grown at a semipermissive temperature they exhibit elevated genomic instability, as indicated by enhanced mitotic gene conversion, mitotic intergenic recombination, chromosomal loss, chromosomal gain, and chromosomal rearrangements. Employing quantitative Southern analysis of chromosomes separated by transverse alternating field gel electrophoresis (TAFE), we have demonstrated that 2N-1 cells monosomic for chromosome VII, owing to the cdc6-1 defect, show slow growth and subsequently yield 2N variants that grow at a normal rate in association with restitution of disomy for chromosome VII. Analysis of TAFE gels also demonstrates that cdc6-1/cdc6-1 diploids give rise to aberrant chromosomes of novel lengths. We propose an explanation for the genomic instability induced by the cdc6-1 mutation, which suggests that hyper-recombination, chromosomal loss, chromosomal gain and chromosomal rearrangements reflect aberrant mitotic division by cdc6-1/cdc6-1 cells containing chromosomes that have not replicated fully.     

The N-terminus of Mcm10 is important for interaction with the 9-1-1 clamp and in resistance to DNA damage

Accurate replication of the genome requires the evolutionarily conserved minichromosome maintenance protein, Mcm10. Although the details of the precise role of Mcm10 in DNA replication are still debated, it interacts with the Mcm2-7 core helicase, the lagging strand polymerase, DNA polymerase-α and the replication clamp, proliferating cell nuclear antigen. Loss of these interactions caused by the depletion of Mcm10 leads to chromosome breakage and cell cycle checkpoint activation. However, whether Mcm10 has an active role in DNA damage prevention is unknown. Here, we present data that establish a novel role of the N-terminus of Mcm10 in resisting DNA damage. We show that Mcm10 interacts with the Mec3 subunit of the 9-1-1 clamp in response to replication stress evoked by UV irradiation or nucleotide shortage. We map the interaction domain with Mec3 within the N-terminal region of Mcm10 and demonstrate that its truncation causes UV light sensitivity. This sensitivity is not further enhanced by a deletion of MEC3, arguing that MCM10 and MEC3 operate in the same pathway. Since Rad53 phosphorylation in response to UV light appears to be normal in N-terminally truncated mcm10 mutants, we propose that Mcm10 may have a role in replication fork restart or DNA repair.     

The Saccharomyces cerevisiae Mcm6/2 and Mcm5/3 ATPase active sites contribute to the function of the putative Mcm2-7 ‘gate’

The Mcm2-7 complex is the eukaryotic replicative helicase, a toroidal AAA⁺ molecular motor that uses adenosine triphosphate (ATP) binding and hydrolysis to separate duplex DNA strands during replication. This heterohexameric helicase contains six different and essential subunits (Mcm2 through Mcm7), with the corresponding dimer interfaces forming ATPase active sites from conserved motifs of adjacent subunits. As all other known hexameric helicases are formed from six identical subunits, the function of the unique heterohexameric organization of Mcm2-7 is of particular interest. Indeed, prior work using mutations in the conserved Walker A box ATPase structural motif strongly suggests that individual ATPase active sites contribute differentially to Mcm2-7 activity. Although only a specific subset of active sites is required for helicase activity, another ATPase active site (Mcm2/5) may serve as a reversible ATP-dependent discontinuity (‘gate’) within the hexameric ring structure. This study analyzes the contribution that two other structural motifs, the Walker B box and arginine finger, make to each Mcm2-7 ATPase active site. Mutational analysis of these motifs not only confirms that Mcm ATPase active sites contribute unequally to activity but implicates the involvement of at least two additional active sites (Mcm5/3 and 6/2) in modulating the activity of the putative Mcm2/5 gate.     

An orc1 allele with a mutated APC motif is female sterile with amplification defects

The origin recognition complex 1 (ORC1) is the largest subunit of the ORC, the heteromeric hexamer. ORC1 is an essential component of the pre-replicative complex (pre-RC) that licenses eukaryote DNA replication origins. The levels of ORC1 fluctuate during the mitotic cell cycle in Drosophila as well as in some human cells. Proteolysis of ORC1 occurs at the end of M phase in Drosophila, which is mediated by the anaphase-promoting complex (APC), and in late S phase in human cells by Skip-Cullin-F box (SCF). Previously we showed that proteolysis of ORC1 by APC is mediated by the ORC1 destruction box (the O-box), an APC motif conserved among species yet distinct from the D-box or KEN-box. Recently we showed that replacing the O-box with the D-box (ORC1^O→D) changes the degradation profile of ORC1 during a canonical cell cycle. Here we report further characterization of the ORC1^O→D allele that turned out to be a useful tool to examine the function of ORC1 in other modes of DNA replication during oogenesis. In endoreplication stages ORC1^O→D does not change any DNA content profiles, consistent with our previous finding that ORC is dispensable for endoreplication. However, in amplification stage replication efficiency of ORC1^O→D is drastically reduced, which resulted in amplification defects that led to thin egg shell phenotype. Taken together, our analyses show that orc1 allele newly identified is female sterile and possesses a unique feature of phenotypes that are distinct in different modes of DNA replication.     

A novel cell-cycle-regulated interaction of the Bloom syndrome helicase BLM with Mcm6 controls replication-linked processes

The Bloom syndrome DNA helicase BLM contributes to chromosome stability through its roles in double-strand break repair by homologous recombination and DNA replication fork restart during the replication stress response. Loss of BLM activity leads to Bloom syndrome, which is characterized by extraordinary cancer risk and small stature. Here, we have analyzed the composition of the BLM complex during unperturbed S-phase and identified a direct physical interaction with the Mcm6 subunit of the minichromosome maintenance (MCM) complex. Using distinct binding sites, BLM interacts with the N-terminal domain of Mcm6 in G1 phase and switches to the C-terminal Cdt1-binding domain of Mcm6 in S-phase, with a third site playing a role for Mcm6 binding after DNA damage. Disruption of Mcm6-binding to BLM in S-phase leads to supra-normal DNA replication speed in unperturbed cells, and the helicase activity of BLM is required for this increased replication speed. Upon disruption of BLM/Mcm6 interaction, repair of replication-dependent DNA double-strand breaks is delayed and cells become hypersensitive to DNA damage and replication stress. Our findings reveal that BLM not only plays a role in the response to DNA damage and replication stress, but that its physical interaction with Mcm6 is required in unperturbed cells, most notably in S-phase as a negative regulator of replication speed.     

The genomic instability of yeast cdc6-1/cdc6-1 mutants involves chromosome structure and recombination

When diploid cells of Saccharomyces cerevisiae homozygous for the temperature-sensitive cell division cycle mutation cdc6-1 are grown at a semipermissive temperature they exhibit elevated genomic instability, as indicated by enhanced mitotic gene conversion, mitotic intergenic recombination, chromosomal loss, chromosomal gain, and chromosomal rearrangements. Employing quantitative Southern analysis of chromosomes separated by transverse alternating field gel electrophoresis (TAFE), we have demonstrated that 2N-1 cells monosomic for chromosome VII, owing to the cdc6-1 defect, show slow growth and subsequently yield 2N variants that grow at a normal rate in association with restitution of disomy for chromosome VII. Analysis of TAFE gels also demonstrates that cdc6-1/cdc6-1 diploids give rise to aberrant chromosomes of novel lengths. We propose an explanation for the genomic instability induced by the cdc6-1 mutation, which suggests that hyper-recombination, chromosomal loss, chromosomal gain and chromosomal rearrangements reflect aberrant mitotic division by cdc6-1/cdc6-1 cells containing chromosomes that have not replicated fully.     

Association of Fission Yeast Orp1 and Mcm6 Proteins with Chromosomal Replication Origins

We have previously shown that replication of fission yeast chromosomes is initiated in distinct regions. Analyses of autonomous replicating sequences have suggested that regions required for replication are very different from those in budding yeast. Here, we present evidence that fission yeast replication origins are specifically associated with proteins that participate in initiation of replication. Most Orp1p, a putative subunit of the fission yeast origin recognition complex (ORC), was found to be associated with chromatin-enriched insoluble components throughout the cell cycle. In contrast, the minichromosome maintenance (Mcm) proteins, SpMcm2p and SpMcm6p, encoded by the nda1(+)/cdc19(+) and mis5(+) genes, respectively, were associated with chromatin DNA only during the G(1) and S phases. Immunostaining of spread nuclei showed SpMcm6p to be localized at discrete foci on chromatin during the G(1) and S phases. A chromatin immunoprecipitation assay demonstrated that Orp1p was preferentially localized at the ars2004 and ars3002 origins of the chromosome throughout the cell cycle, while SpMcm6p was associated with these origins only in the G(1) and S phases. Both Orp1p and SpMcm6p were associated with a 1-kb region that contains elements required for autonomous replication of ars2004. The results suggest that the fission yeast ORC specifically interacts with chromosomal replication origins and that Mcm proteins are loaded onto the origins to play a role in initiation of replication.     

Characterization of the interaction of GABARAPL-1 with the LIR motif of NBR1

Selective autophagy requires the specific segregation of targeted proteins into autophagosomes. The selectivity is mediated by autophagy receptors, such as p62 and NBR1, which can bind to autophagic effector proteins (Atg8 in yeast, MAP1LC3 protein family in mammals) anchored in the membrane of autophagosomes. Recognition of autophagy receptors by autophagy effectors takes place through an LC3 interaction region (LIR). The canonical LIR motif consists of a WXXL sequence, N-terminally preceded by negatively charged residues. The LIR motif of NBR1 presents differences to this classical LIR motif with a tyrosine residue and an isoleucine residue substituting the tryptophan residue and the leucine residue, respectively. We have determined the structure of the GABARAPL-1/NBR1-LIR complex and studied the influence of the different residues belonging to the LIR motif for the interaction with several mammalian autophagy modifiers (LC3B and GABARAPL-1). Our results indicate that the presence of a tryptophan residue in the LIR motif increases the binding affinity. Substitution by other aromatic amino acids or increasing the number of negatively charged residues at the N-terminus of the LIR motif, however, has little effect on the binding affinity due to enthalpy-entropy compensation. This indicates that different LIRs can interact with autophagy modifiers with unique binding properties.     

Analysis of Mcm2-7 chromatin binding during anaphase and in the transition to quiescence in fission yeast

Mcm2-7 proteins are generally considered to function as a heterohexameric complex, providing helicase activity for the elongation step of DNA replication. These proteins are loaded onto replication origins in M-G1 phase in a process termed licensing or pre-replicative complex formation. It is likely that Mcm2-7 proteins are loaded onto chromatin simultaneously as a pre-formed hexamer although some studies suggest that subcomplexes are recruited sequentially. To analyze this process in fission yeast, we have compared the levels and chromatin binding of Mcm2-7 proteins during the fission yeast cell cycle. Mcm subunits are present at approximately 1 x 10(4) molecules/cell and are bound with approximately equal stoichiometry on chromatin in G1/S phase cells. Using a single cell assay, we have correlated the timing of chromatin association of individual Mcm subunits with progression through mitosis. This showed that Mcm2, 4 and 7 associate with chromatin at about the same stage of anaphase, suggesting that licensing involves the simultaneous binding of these subunits. We also examined Mcm2-7 chromatin association when cells enter a G0-like quiescent state. Chromatin binding is lost in this transition in a process that does not require DNA replication or the selective degradation of specific subunits.     

Modulation of CD6 function through interaction with Galectin-1 and -3

CD6 is a lymphocyte glycoprotein receptor that physically associates with the antigen-specific receptor complex at the center of the immunological synapse, where it interacts with its ligand CD166/ALCAM. The present work reports the carbohydrate-dependent interaction of CD6 and CD166/ALCAM with Galectin-1 and -3, two well-known soluble mammalian lectins. Both galectins interfered with superantigen-induced T cell proliferation and cell adhesion phenomena mediated by the CD6-CD166/ALCAM pair, while CD6 expression protected cells from galectin-induced apoptosis. The results suggest that interaction of Galectin-1 and -3 with CD6 and CD166/ALCAM might modulate some relevant aspects of T cell physiology.     

Roles of chromatin assembly factor 1 in the epigenetic control of chromatin plasticity

Genetic information embedded in DNA sequence and the epigenetic information marked by modifications on DNA and histones are essential for the life of eukaryotes. Cells have evolved mechanisms of DNA duplication and chromatin restoration to ensure the inheritance of genetic and epigenetic information during cell division and development. In this review, we focus on the maintenance of epigenetic landscape during chromatin dynamics which requires the orchestration of histones and their chaperones. We discuss how epigenetic marks are re-established in the assembly of new chromatin after DNA replication and repair, highlighting the roles of CAF-1 in the process of changing chromatin state. The functions of CAF-1 provide a link between chromatin assembly and epigenetic restoration.     

Prothymosin alpha modulates the interaction of histone H1 with chromatin.

Prothymosin alpha (ProTalpha) is an abundant acidic nuclear protein that may be involved in cell proliferation. In our search for its cellular partners, we have recently found that ProTalpha binds to linker histone H1. We now provide further evidence for the physiological relevance of this interaction by immunoisolation of a histone H1-ProTalpha complex from NIH 3T3 cell extracts. A detailed analysis of the interaction between the two proteins suggests contacts between the acidic region of ProTalpha and histone H1. In the context of a physiological chromatin reconstitution reaction, the presence of ProTalpha does not affect incorporation of an amount of histone H1 sufficient to increase the nucleosome repeat length by 20 bp, but prevents association of all further H1. Consistent with this finding, a fraction of histone H1 is released when H1-containing chromatin is challenged with ProTalpha. These results imply at least two different interaction modes of H1 with chromatin, which can be distinguished by their sensitivity to ProTalpha. The properties of ProTalpha suggest a role in fine tuning the stoichiometry and/or mode of interaction of H1 with chromatin.     

Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2

Chromatin assembly factor 1 (CAF1) consisting of p150, p60 and p48 is known to assemble histones onto newly synthesized DNA and thus maintain the chromatin structure. Here, we show that CAF1 expression was induced in human cytomegalovirus (HCMV)-infected cells, concomitantly with global chromatin decondensation. This apparent conflict was thought to result, in part, from CAF1 mislocalization to compartments of HCMV DNA synthesis through binding of its largest subunit p150 to viral immediate-early protein 2 (IE2). p150 interaction with p60 and IE2 facilitated HCMV DNA synthesis. The IE2Q548R mutation, previously reported to result in impaired HCMV growth with unknown mechanism, disrupted IE2/p150 and IE2/histones association in our study. Moreover, IE2 interaction with histones partly depends on p150, and the HCMV-induced chromatin decondensation was reduced in cells ectopically expressing the p150 mutant defective in IE2 binding. These results not only indicate that CAF1 was hijacked by IE2 to facilitate the replication of the HCMV genome, suggesting chromatin assembly plays an important role in herpesviral DNA synthesis, but also provide a model of the virus-induced chromatin instability through CAF1.     

Roles of Mcm7 and Mcm4 subunits in the DNA helicase activity of the mouse Mcm4/6/7 complex

Mcm, which is composed of six structurally related subunits (Mcm2-7), is essential for eukaryotic DNA replication. A subassembly of Mcm, the Mcm4/6/7 double-trimeric complex, possesses DNA helicase activity, and it has been proposed that Mcm may function as a replicative helicase at replication forks. We show here that conserved ATPase motifs of Mcm7 are essential for ATPase and DNA helicase activities of the Mcm4/6/7 complex. Because uncomplexed Mcm7 displayed neither ATPase nor DNA helicase activity, Mcm7 contributes to the DNA helicase activity of the Mcm complex through interaction with other subunits. In contrast, the Mcm4/6/7 complex containing a zinc finger mutant of Mcm4 with partially impaired DNA binding activity exhibited elevated DNA helicase activity. The Mcm4/6/7 complex containing this Mcm4 mutant tended to dissociate into trimeric complexes, suggesting that the zinc finger of Mcm4 is involved in subunit interactions of trimers. The Mcm4 mutants lacking the N-terminal 35 or 112 amino acids could form hexameric Mcm4/6/7 complexes, but displayed very little DNA helicase activity. In conjunction with the previously reported essential role of Mcm6 in ATP binding (You, Z., Komamura, Y., and Ishimi, Y. (1999) Mol. Cell. Biol. 19, 8003-8015), our data indicate distinct roles of Mcm4, Mcm6, and Mcm7 subunits in activation of the DNA helicase activity of the Mcm4/6/7 complex.     

Structure of Sla1p homology domain 1 and interaction with the NPFxD endocytic internalization motif

Adaptor proteins play important endocytic roles including recognition of internalization signals in transmembrane cargo. Sla1p serves as the adaptor for uptake of transmembrane proteins containing the NPFxD internalization signal, and is essential for normal functioning of the actin cytoskeleton during endocytosis. The Sla1p homology domain 1 (SHD1) within Sla1p is responsible for recognition of the NPFxD signal. This study presents the NMR structure of the NPFxD-bound state of SHD1 and a model for the protein-ligand complex. The alpha+beta structure of the protein reveals an SH3-like topology with a solvent-exposed hydrophobic ligand binding site. NMR chemical shift perturbations and effects of structure-based mutations on ligand binding in vitro define residues that are key for NPFxD binding. Mutations that abolish ligand recognition in vitro also abolish NPFxD-mediated receptor internalization in vivo. Thus, SHD1 is a novel functional domain based on SH3-like topology, which employs a unique binding site to recognize the NPFxD endocytic internalization signal. Its distant relationship with the SH3 fold endows this superfamily with a new role in endocytosis.