Rvb1p/Rvb2p recruit Arp5p and assemble a functional Ino80 chromatin remodeling complex

The Rvb1p and Rvb2p (or TIP48 and TIP49) nuclear ATP binding proteins are universally conserved in eukaryotes and essential for viability of yeasts. Rvbp associate with each other as a double hexamer, with YHR034c and with two complexes involved in chromatin remodeling, Ino80.com and Swr1.com. Loss of Rvb1p or Ino80p affects many yeast promoters similarly. Rvbp are not essential for the recruitment of Ino80p to promoters but are essential for the catalytic activity of Ino80.com. Loss of Rvbp leads to loss of the functionally critical Arp5p in Ino80.com. Rvb2p associates with Arp5p in vitro in a reaction dependent on the presence of ATP and Ino80p. Therefore, Rvbp are required for the structural and functional integrity of the Ino80 chromatin remodeling complex.     

Plasmodium falciparum RuvB1 is an active DNA helicase and translocates in the 5′–3′ direction

RuvB family of protein contains two similar kinds of proteins i.e. RuvB1 and RuvB2 from yeast to human. These proteins belong to the AAA + class of proteins and are critical components of several multiprotein complexes involved in diverse cellular activities. There are two RuvB proteins annotated in the Plasmodium database but the identification of the third protein recently by our lab has raised the question why Plasmodium falciparum contains three RuvB proteins instead of two. Hence the biochemical characterizations of these proteins have become essential to understand the role of these proteins in the malaria parasite. Recently we have reported the characterization of the recombinant PfRuvB3, which contains ATPase activity but lacks DNA helicase activity. In the present study we report the phylogenetic analysis and detailed biochemical characterization of one of the other RuvB homologue RuvB1 from P. falciparum. PfRuvB1 shows considerable homology with human as well as yeast RuvB1 and contains Walker motif A and Walker motif B. The activity analysis of this protein revealed that PfRuvB1 is an ATPase and this activity increased significantly in the presence of ss-DNA. PfRuvB1 also contains DNA helicase activity and translocates preferentially in 5′ to 3′ direction. In vivo investigation of PfRuvB1 revealed that it is constitutively expressed during all the stages of intraerythrocytic cycle of P. falciparum and localizes mainly to the nucleus. These studies will make important contribution in understanding the role of RuvB protein in P. falciparum.     

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.     

The Combination of X-Ray Crystallography and Cryo-Electron Microscopy Provides Insight into the Overall Architecture of the Dodecameric Rvb1/Rvb2 Complex

The Rvb1/Rvb2 complex is an essential component of many cellular pathways. The Rvb1/Rvb2 complex forms a dodecameric assembly where six copies of each subunit form two heterohexameric rings. However, due to conformational variability, the way the two rings pack together is still not fully understood. Here, we present the crystal structure and two cryo-electron microscopy reconstructions of the dodecameric, full-length Rvb1/Rvb2 complex, all showing that the interaction between the two heterohexameric rings is mediated through the Rvb1/Rvb2-specific domain II. Two conformations of the Rvb1/Rvb2 dodecamer are present in solution: a stretched conformation also present in the crystal, and a compact conformation. Novel asymmetric features observed in the reconstruction of the compact conformation provide additional insight into the plasticity of the Rvb1/Rvb2 complex.     

Functional and comparative characterization of Saccharomyces cerevisiae RVB1 and RVB2 genes with bacterial Ruv homologues

Expression of yeast RuvB-like gene analogues of bacterial RuvB is self-regulated, as episomal overexpression of RVB1 and RVB2 decreases the expression of their chromosomal copies by 85%. Heterozygosity for either gene correlates with lower double-strand break repair of inverted-repeat DNA and decreased survival after UV irradiation, suggesting their haploinsufficiency, while overexpression of the bacterial RuvAB complex improves UV survival in yeast. Rvb2p preferentially binds artificial DNA Holiday junctions like the bacterial RuvAB complex, whereas Rvb1p binds to duplex or cruciform DNA. As both proteins also interact with chromatin, their role in recombination and repair through chromatin remodelling, and their evolutionary relationship to the bacterial homologue, is discussed.     

Plasmodium falciparum RuvB2 translocates in 5′–3′ direction,relocalizes during schizont stage and its enzymatic activities are up regulated by RuvB3 of the same complex

Two similar proteins RuvB like1 (Rvb1/Pontin) and RuvB like2 (Rvb2/Reptin) of AAA + family of enzymes are present in yeast to human and are well known to be involved in diverse cellular activities. The human malaria parasite Plasmodium falciparum contains three different RuvB like proteins. Thus it has been of interest to explore why P. falciparum requires three RuvB like proteins and how these enzymes are biochemically regulated. In this study, we present the detailed biochemical characterization of PfRuvB2. The complex of PfRuvB3 was immunopurified and the presence of PfRuvB2 was confirmed. The in vitro interaction study shows that PfRuvB2 interacts only with PfRuvB3 but not with PfRuvB1. The recombinant as well as endogenous PfRuvB2 contains ATPase as well as weak DNA helicase activities. The presence of PfRuvB3 in the helicase reaction of PfRuvB2 increases the helicase activity significantly. Interestingly PfRuvB2/PfRuvB3 complex preferentially translocates and unwinds DNA in the 5′–3′ direction. In vivo studies showed that PfRuvB2 is expressed in all the asexual intraerythrocytic developmental stages and localizes mainly in the nucleus during merozoite, ring and trophozoite stages while during schizont stage it relocalizes partially in the nucleus and partially towards cytoplasm. As PfRuvB3 is specific to intraerythrocytic mitosis so we interpret that PfPuvB3 interacts with PfRuvB2 during schizont/intraerythrocytic mitosis and acts as its modulator mainly for the appreciable helicase activity.     

A mammalian chromatin remodeling complex with similarities to the yeast INO80 complex

The mammalian Tip49a and Tip49b proteins belong to an evolutionarily conserved family of AAA+ ATPases. In Saccharomyces cerevisiae, orthologs of Tip49a and Tip49b, called Rvb1 and Rvb2, respectively, are subunits of two distinct ATP-dependent chromatin remodeling complexes, SWR1 and INO80. We recently demonstrated that the mammalian Tip49a and Tip49b proteins are integral subunits of a chromatin remodeling complex bearing striking similarities to the S. cerevisiae SWR1 complex (Cai, Y., Jin, J., Florens, L., Swanson, S. K., Kusch, T., Li, B., Workman, J. L., Washburn, M. P., Conaway, R. C., and Conaway, J. W. (2005) J. Biol. Chem. 280, 13665-13670). In this report, we identify a new mammalian Tip49a- and Tip49b-containing ATP-dependent chromatin remodeling complex, which includes orthologs of 8 of the 15 subunits of the S. cerevisiae INO80 chromatin remodeling complex as well as at least five additional subunits unique to the human INO80 (hINO80) complex. Finally, we demonstrate that, similar to the yeast INO80 complex, the hINO80 complex exhibits DNA- and nucleosome-activated ATPase activity and catalyzes ATP-dependent nucleosome sliding.     

The dosage of chromatin proteins affects transcriptional silencing and DNA repair in Saccharomyces cerevisiae

Alterations in protein composition or dosage within chromatin may trigger changes in processes such as gene expression and DNA repair. Through transposon mutagenesis and targeted gene deletions in haploids and diploids of Saccharomyces cerevisiae, we identified mutations that affect telomeric silencing in genes encoding telomere-associated Sir4p and Yku80p and chromatin remodeling ATPases Ies2p and Rsc1p. We found that sir4/SIR4 heterozygous diploids efficiently silence the mating type locus HMR but not telomeres, and diploids heterozygous for yku80 and ies2 mutations are inefficient at DNA repair. In contrast, strains heterozygous for most chromatin remodeling ATPase mutations retain wild-type silencing and DNA repair levels. Thus, in diploids, chromatin structures required for DNA repair and telomeric silencing are sensitive to dosage changes.     

How to load a replicative helicase onto chromatin: A more and more complex matter during evolution

In all eukaryotes, the heterohexameric MCM2-7 complex functions as the main replicative helicase during S phase. During early G1 phase, it is recruited onto chromatin in a sequence of reactions called pre-replication complex (pre-RC) formation or DNA licensing. This process is ATP-dependent and at least two different chromatin-bound ATPase activities are required besides several others essential, but not enzymatically active, proteins. Although functionally conserved during evolution, pre-RC formation and the way the MCM2-7 helicase is loaded onto DNA are more complex in metazoans than in single-cell eukaryotes. Recently, we characterized a new essential factor for pre-RC assembly and DNA licensing, the vertebrate-specific MCM9 protein that contains not only an ATPase but also a helicase domain. MCM9 adds another layer of complexity to how vertebrates achieve and regulate the loading of the MCM2-7 helicase and DNA replication.     

The ATPase Cycle of PcrA Helicase and Its Coupling to Translocation on DNA

The superfamily 1 bacterial helicase PcrA has a role in the replication of certain plasmids, acting with the initiator protein (RepD) that binds to and nicks the double-stranded origin of replication. PcrA also translocates single-stranded DNA with discrete steps of one base per ATP hydrolyzed. Individual rate constants have been determined for the DNA helicase PcrA ATPase cycle when bound to either single-stranded DNA or a double-stranded DNA junction that also has RepD bound. The fluorescent ATP analogue 2′(3′)-O-(N-methylanthraniloyl)ATP was used throughout all experiments to provide a complete ATPase cycle for a single nucleotide species. Fluorescence intensity and anisotropy stopped-flow measurements were used to determine rate constants for binding and release. Quenched-flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor MDCC-PBP was used to measure phosphate release kinetics. The chemical cleavage step is the rate-limiting step in the cycle and is essentially irreversible and would result in the bound ATP complex being a major component at steady state. This cleavage step is greatly accelerated by bound DNA, producing the high activation of this protein compared to the protein alone. The data suggest the possibility that ADP is released in two steps, which would result in bound ADP also being a major intermediate, with bound ADP·P_i being a very small component. It therefore seems likely that the major transition in structure occurs during the cleavage step, rather than P_i release. ATP rebinding could then cause reversal of this structural transition. The kinetic mechanism of the PcrA ATPase cycle is very little changed by potential binding to RepD, supporting the idea that RepD increases the processivity of PcrA by increasing affinity to DNA rather than affecting the enzymatic properties per se.