Structure-function analysis of the RNA helicase maleless

Loss of function of the RNA helicase maleless (MLE) in Drosophila melanogaster leads to male-specific lethality due to a failure of X chromosome dosage compensation. MLE is presumably involved in incorporating the non-coding roX RNA into the dosage compensation complex (DCC), which is an essential but poorly understood requirement for faithful targeting of the complex to the X chromosome. Sequence comparison predicts several RNA-binding domains in MLE but their properties have not been experimentally verified. We evaluated the RNA-binding characteristics of these conserved motifs and their contributions to RNA-stimulated ATPase activity, to helicase activity, as well as to the targeting of MLE to the nucleus and to the X chromosome territory. We find that RB2 is the dominant, conditional RNA-binding module, which is indispensable for ATPase and helicase activity whereas the N-terminal RB1 motif does not bind RNA, but is involved in targeting MLE to the X chromosome. The C-terminal domain containing a glycine-rich heptad repeat adds potential dimerization and RNA-binding surfaces which are not required for helicase activity.     

The NTPase/helicase activities of Drosophila maleless, an essential factor in dosage compensation.

Drosophila maleless (mle) is required for X chromosome dosage compensation and is essential for male viability. Maleless protein (MLE) is highly homologous to human RNA helicase A and the bovine counterpart of RNA helicase A, nuclear helicase II. In this report, we demonstrate that MLE protein, overexpressed and purified from Sf9 cells infected with recombinant baculovirus, possesses RNA/DNA helicase, adenosine triphosphatase (ATPase) and single-stranded (ss) RNA/ssDNA binding activities, properties identical to RNA helicase A. Using site-directed mutagenesis, we created a mutant of MLE (mle-GET) that contains a glutamic acid in place of lysine in the conserved ATP binding site A. In vitro biochemical analysis showed that this mutation abolished both NTPase and helicase activities of MLE but affected the ability of MLE to bind to ssRNA, ssDNA and guanosine triphosphate (GTP) less severely. In vivo, mle-GET protein could still localize to the male X chromosome coincidentally with the male-specific lethal-1 protein, MSL-1, but failed to complement mle1 mutant males. These results indicate that the NTPase/helicase activities are essential functions of MLE for dosage compensation, perhaps utilized for chromatin remodeling of X-linked genes.     

Targeting the chromatin-remodeling MSL complex of Drosophila to its sites of action on the X chromosome requires both acetyl transferase and ATPase activities

Dosage compensation in Drosophila is mediated by a multiprotein, RNA-containing complex that associates with the X chromosome at multiple sites. We have investigated the role that the enzymatic activities of two complex components, the histone acetyltransferase activity of MOF and the ATPase activity of MLE, may have in the targeting and association of the complex with the X chromosome. Here we report that MLE and MOF activities are necessary for complexes to access the various X chromosome sites. The role that histone H4 acetylation plays in this process is supported by our observations that MOF overexpression leads to the ectopic association of the complex with autosomal sites.     

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.     

Characterization of the enzymatic activity of hChlR1, a novel human DNA helicase

Recently, we cloned two highly related human genes, hChlR1 (DDX11) and hChlR2 (DDX12), which appear to be homologs of the Saccharomyces cerevisiae CHL1 gene. Nucleotide sequence analysis suggests that these genes encode new members of the DEAH family of DNA helicases. While the enzymatic activity of CHL1 has not been characterized, the protein is required for the maintenance of high fidelity chromosome segregation in yeast. Here we report that the hChlR1 protein is a novel human DNA helicase. We have expressed and purified hChlR1 using a baculovirus system and analyzed its enzymatic activity. The recombinant hChlR1 protein possesses both ATPase and DNA helicase activities that are strictly dependent on DNA, divalent cations and ATP. These activities are abolished by a single amino acid substitution in the ATP-binding domain. The hChlR1 protein can unwind both DNA/DNA and RNA/DNA substrates. It has a preference for movement in the 5′→3′ direction on short single-stranded DNA templates. However, unlike other DNA helicases, the hChlR1 DNA helicase can translocate along single-stranded DNA in both directions when substrates have a very long single-stranded DNA region. The enzymatic activities of hChlR1 suggest that DNA helicases are required for maintaining the fidelity of chromosome segregation.     

Translocation by the RecB motor is an absolute requirement for {chi}-recognition and RecA protein loading by RecBCD enzyme

RecBCD enzyme is a heterotrimeric helicase/nuclease that initiates homologous recombination at double-stranded DNA breaks. The enzyme is driven by two motor subunits, RecB and RecD, translocating on opposite single-strands of the DNA duplex. Here we provide evidence that, although both motor subunits can support the translocation activity for the enzyme, the activity of the RecB subunit is necessary for proper function of the enzyme both in vivo and in vitro. We demonstrate that the RecBCD(K177Q) enzyme, in which RecD helicase is disabled by mutation of the ATPase active site, complements recBCD deletion in vivo and displays all of the enzymatic activities that are characteristic of the wild-type enzyme in vitro. These include helicase and nuclease activities and the abilities to recognize the recombination hotspot chi and to coordinate the loading of RecA protein onto the ssDNA it produces. In contrast, the RecB(K29Q)CD enzyme, carrying a mutation in the ATPase site of RecB helicase, fails to complement recBCD deletion in vivo. We further show that even though RecB(K29Q)CD enzyme displays helicase and nuclease activities, its inability to translocate along the 3'-terminated strand results in the failure to recognize chi and to load RecA protein. Our findings argue that translocation by the RecB motor is required to deliver RecC subunit to chi, whereas the RecD subunit has a dispensable motor activity but an indispensable regulatory function.     

Modulation of Heterochromatin by Male Specific Lethal Proteins and roX RNA in Drosophila melanogaster Males

The ribonucleoprotein Male Specific Lethal (MSL) complex is required for X chromosome dosage compensation in Drosophila melanogaster males. Beginning at 3 h of development the MSL complex binds transcribed X-linked genes and modifies chromatin. A subset of MSL complex proteins, including MSL1 and MSL3, is also necessary for full expression of autosomal heterochromatic genes in males, but not females. Loss of the non-coding roX RNAs, essential components of the MSL complex, lowers the expression of heterochromatic genes and suppresses position effect variegation (PEV) only in males, revealing a sex-limited disruption of heterochromatin. To explore the molecular basis of this observation we examined additional proteins that participate in compensation and found that MLE, but not Jil-1 kinase, contributes to heterochromatic gene expression. To determine if identical regions of roX RNA are required for dosage compensation and heterochromatic silencing, we tested a panel of roX1 transgenes and deletions and find that the X chromosome and heterochromatin functions are separable by some mutations. Chromatin immunoprecipitation of staged embryos revealed widespread autosomal binding of MSL3 before and after localization of the MSL complex to the X chromosome at 3 h AEL. Autosomal MSL3 binding was dependent on MSL1, supporting the idea that a subset of MSL proteins associates with chromatin throughout the genome during early development. The broad localization of these proteins early in embryogenesis supports the idea of direct action at autosomal sites. We postulate that this may contribute to the sex-specific differences in heterochromatin that we, and others, have noted.     

Multiple classes of MSL binding sites target dosage compensation to the X chromosome of Drosophila

MSL complexes bind hundreds of sites along the single male X chromosome to achieve dosage compensation in Drosophila. Previously, we proposed that approximately 35 "high-affinity" or "chromatin entry" sites (CES) might nucleate spreading of MSL complexes in cis to paint the X chromosome. This was based on analysis of the first characterized sites roX1 and roX2. roX transgenes attract MSL complex to autosomal locations where it can spread long distances into flanking chromatin. roX1 and roX2 also produce noncoding RNA components of the complex. Here we identify a third site from the 18D10 region of the X chromosome. Like roX genes, 18D binds full and partial MSL complexes in vivo and encompasses a male-specific DNase I hypersensitive site (DHS). Unlike roX genes, the 510 bp 18D site is apparently not transcribed and shows high affinity for MSL complex and spreading only as a multimer. While mapping 18D, we discovered MSL binding to X cosmids that do not carry one of the approximately 35 high-affinity sites. Based on additional analyses of chromosomal transpositions, we conclude that spreading in cis from the roX genes or the approximately 35 originally proposed "entry sites" cannot be the sole mechanism for MSL targeting to the X chromosome.     

Intermolecular interactions within the abundant DEAD-box protein Dhh1 regulate its activity in vivo

Dhh1 is a highly conserved DEAD-box protein that has been implicated in many processes involved in mRNA regulation. At least some functions of Dhh1 may be carried out in cytoplasmic foci called processing bodies (P-bodies). Dhh1 was identified initially as a putative RNA helicase based solely on the presence of conserved helicase motifs found in the superfamily 2 (Sf2) of DEXD/H-box proteins. Although initial mutagenesis studies revealed that the signature DEAD-box motif is required for Dhh1 function in vivo, enzymatic (ATPase or helicase) or ATP binding activities of Dhh1 or those of any its many higher eukaryotic orthologues have not been described. Here we provide the first characterization of the biochemical activities of Dhh1. Dhh1 has weaker RNA-dependent ATPase activity than other well characterized DEAD-box helicases. We provide evidence that intermolecular interactions between the N- and C-terminal RecA-like helicase domains restrict its ATPase activity; mutation of residues mediating these interactions enhanced ATP hydrolysis. Interestingly, the interdomain interaction mutant displayed enhanced mRNA turnover, RNA binding, and recruitment into cytoplasmic foci in vivo compared with wild type Dhh1. Also, we demonstrate that the ATPase activity of Dhh1 is not required for it to be recruited into cytoplasmic foci, but it regulates its association with RNA in vivo. We hypothesize that the activity of Dhh1 is restricted by interdomain interactions, which can be regulated by cellular factors to impart stringent control over this very abundant RNA helicase.     

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.