The pentapeptide motif of Hox proteins is required for cooperative DNA binding with Pbx1, physically contacts Pbx1, and enhances DNA binding by Pbx1.

The vertebrate Hox genes, which represent a subset of all homeobox genes, encode proteins that regulate anterior-posterior positional identity during embryogenesis and are cognates of the Drosophila homeodomain proteins encoded by genes composing the homeotic complex (HOM-C). Recently, we demonstrated that multiple Hox proteins bind DNA cooperatively with both Pbx1 and its oncogenic derivative, E2A-Pbx1. Here, we show that the highly conserved pentapeptide motif F/Y-P-W-M-R/K, which occurs in numerous Hox proteins and is positioned 8 to 50 amino acids N terminal to the homeodomain, is essential for cooperative DNA binding with Pbx1 and E2A-Pbx1. Point mutational analysis demonstrated that the tryptophan and methionine residues within the core of this motif were critical for cooperative DNA binding. A peptide containing the wild-type pentapeptide sequence, but not one in which phenylalanine was substituted for tryptophan, blocked the ability of Hox proteins to bind cooperatively with Pbx1 or E2A-Pbx1, suggesting that the pentapeptide itself provides at least one surface through which Hox proteins bind Pbx1. Furthermore, the same peptide, but not the mutant peptide, stimulated DNA binding by Pbx1, suggesting that interaction of Hox proteins with Pbx1 through the pentapeptide motif raises the DNA-binding ability of Pbx1.     

Hoxa9 transforms primary bone marrow cells through specific collaboration with Meis1a but not Pbx1b.

Hoxa9, Meis1 and Pbx1 encode homeodomaincontaining proteins implicated in leukemic transformation in both mice and humans. Hoxa9, Meis1 and Pbx1 proteins have been shown to physically interact with each other, as Hoxa9 cooperatively binds consensus DNA sequences with Meis1 and with Pbx1, while Meis1 and Pbx1 form heterodimers in both the presence and absence of DNA. In this study, we sought to determine if Hoxa9 could transform hemopoietic cells in collaboration with either Pbx1 or Meis1. Primary bone marrow cells, retrovirally engineered to overexpress Hoxa9 and Meis1a simultaneously, induced growth factor-dependent oligoclonal acute myeloid leukemia in <3 months when transplanted into syngenic mice. In contrast, overexpression of Hoxa9, Meis1a or Pbx1b alone, or the combination of Hoxa9 and Pbx1b failed to transform these cells acutely within 6 months post-transplantation. Similar results were obtained when FDC-P1 cells, engineered to overexpress these genes, were transplanted to syngenic recipients. Thus, these studies demonstrate a selective collaboration between a member of the Hox family and one of its DNA-binding partners in transformation of hemopoietic cells.     

Glutathione-S-transferases are major cytosolic thyroid hormone binding proteins

Thyroid hormone binding proteins of rat liver cytosol were characterized. Glutathione-S-transferases were identified among major cytosolic proteins adsorbed by thyroxine affinity matrices. The Ya and Yb subunits of the glutathione-S-transferases were also principal proteins of cytosol covalently labeled with 3,3',5-triiodo-L-thyronine (T3) or 3,3',5,5'-tetraiodo-L-thyronine (T4) by photoaffinity methods. T3 and T4, but not L-thyronine or iodinated tyrosines, were bound with high affinity to purified glutathione-S-transferases and were potent inhibitors of their enzymatic activities. These results suggest that glutathione-S-transferases have the potential to function in the intracellular binding and transport of thyroid hormones. The proteins provide a means for regulating the action and metabolism of thyroid hormones by acting as high capacity binding components.     

Structural determinants within Pbx1 that mediate cooperative DNA binding with pentapeptide-containing Hox proteins: proposal for a model of a Pbx1-Hox-DNA complex.

Genetic studies have identified a family of divergent homeodomain proteins, including the human protooncoprotein Pbx1 and its drosophila homolog extradenticle (Exd), which function as cofactors with a subset of Hox and HOM-C proteins, and are essential for specific target gene expression. Pbx1/Exd binds DNA elements cooperatively with a large subset of Hox/HOM-C proteins containing a conserved pentapeptide motif, usually YPWMR, located just N terminally to their homeodomains. The pentapeptide is essential for cooperative DNA binding with Pbx1. In this study, we identify structural determinants of Pbx1 that are required for cooperative DNA binding with the pentapeptide-containing Hox protein HoxA5. We demonstrate that the homeodomain of Pbx1 contains a surface that binds the pentapeptide motif and that the Pbx1 homeodomain is sufficient for cooperative DNA binding with a Hox protein. A sequence immediately C terminal to the Pbx1 homeodomain, which is highly conserved in Pbx2 and Pbx3 and predicted to form an alpha-helix, enhances monomeric DNA binding by Pbx1 and also contributes to maximal cooperativity with Hox proteins. Binding studies with chimeric HoxA5-Pbx1 fusion proteins suggest that the homeodomains of Pbx1 and HoxA5 are docked on the representative element, TTGATTGAT, in tandem, with Pbx1 recognizing the 5' TTGAT core motif and the Hox protein recognizing the 3' TGAT core. The proposed binding orientation permits Hox proteins to exhibit further binding specificity on the basis of the identity of the four residues 3' to their core binding motif.     

Tallimustine lesions in cellular DNA are AT sequence-specific but not region-specific.

Tallimustine (FCE 24517) is an AT-specific alkylating antitumor derivative of distamycin. This study examined levels of tallimustine lesions in intracellular DNA, their sequence- and region-specificity, and the long-range distribution of the drug binding motif. Tallimustine adducts in DNA converted to strand breaks by heating allowed the quantitation of drug lesions. In bulk DNA of intact human leukemia CEM cells, tallimustine formed 0.15 +/- 0.04 and 0.64 +/- 0.18 lesions/kbp at 5 and 50 microM, respectively. These lesions represent monoadducts as no interstrand cross-links or DNA-protein cross-links were detected. Tallimustine adducts in intracellularly treated DNA showed a general preference for sequences with T-tracts, suggesting a propensity for intrinsically bent motifs. Major drug-adducted sites identified by repetitive primer extension, included 5'-TTTTGPu-3' and 5'-TTTTGC-3' motif. Despite the high specificity at the nucleotide level, tallimustine did not differentiate among bulk DNA and three discrete AT-rich regions of genomic DNA examined by quantitative PCR stop assay with lesion frequencies ranging from 0.23 to 0.39 lesions/kbp at 25 microM drug. In comparisons of lesion frequencies and cytotoxicity, tallimustine adducts are approximately 50 times more lethal than relatively nonsequence specific cisplatin adducts but are >100 times less lethal than lesions by an unrelated AT-specific drug, bizelesin. However, the 5'-TTTTGPu-3' motifs targeted by tallimustine are relatively infrequent and scattered throughout the genome. In contrast, the motifs 5'-T(A/T)(4)A-3' motifs targeted by bizelesin, while also infrequent, cluster in defined AT-rich islands. The lack of region-specificity may be the reason tallimustine adducts, despite high AT-specificity at the nucleotide level, are less lethal than region-specific bizelesin adducts.     

Motifs Q and I are required for ATP hydrolysis but not for ATP binding in SWI2/SNF2 proteins

Active DNA-dependent ATPase A Domain (ADAAD) is a SWI2/SNF2 protein that hydrolyzes ATP in the presence of stem-loop DNA that contains both double-stranded and single-stranded regions. ADAAD possesses the seven helicase motifs that are a characteristic feature of all the SWI2/SNF2 proteins present in yeast as well as mammalian cells. In addition, these proteins also possess the Q motif ~17 nucleotides upstream of motif I. Using site-directed mutagenesis, we have sought to define the role of motifs Q and I in ATP hydrolysis mediated by ADAAD. We show that in ADAAD both motifs Q and I are required for ATP catalysis but not for ATP binding. In addition, the conserved glutamine present in motif Q also dictates the catalytic rate. The ability of the conserved glutamine present in motif Q to dictate the catalytic rate has not been observed in helicases. Further, the SWI2/SNF2 proteins contain a conserved glutamine, one amino acid residue downstream of motif I. This conserved glutamine, Q244 in ADAAD, also directs the rate of catalysis but is not required either for hydrolysis or for ligand binding. Finally, we show that the adenine moiety of ATP is sufficient for interaction with SWI2/SNF2 proteins. The γ-phosphate of ATP is required for inducing the conformational change that leads to ATPase activity. Thus, the SWI2/SNF2 proteins despite sequence conservation with helicases appear to behave in a manner distinct from that of the helicases.     

DNA binding is not sufficient for nuclear localization of regulatory proteins in Saccharomyces cerevisiae.

We showed by immunofluorescence that the procaryotic DNA-binding protein LexA and a chimeric protein that contains the DNA-binding portion of LexA (amino acids 1 to 87) and a large portion (amino acids 74 to 881) of the Saccharomyces cerevisiae positive regulatory GAL4 protein (GAL4 gene product) are not preferentially localized in the nucleus in S. cerevisiae.     

The major magnetosome proteins MamGFDC are not essential for magnetite biomineralization in Magnetospirillum gryphiswaldense but regulate the size of magnetosome crystals

Magnetospirillum gryphiswaldense and related magnetotactic bacteria form magnetosomes, which are membrane-enclosed organelles containing crystals of magnetite (Fe₃O₄) that cause the cells to orient in magnetic fields. The characteristic sizes, morphologies, and patterns of alignment of magnetite crystals are controlled by vesicles formed of the magnetosome membrane (MM), which contains a number of specific proteins whose precise roles in magnetosome formation have remained largely elusive. Here, we report on a functional analysis of the small hydrophobic MamGFDC proteins, which altogether account for nearly 35% of all proteins associated with the MM. Although their high levels of abundance and conservation among magnetotactic bacteria had suggested a major role in magnetosome formation, we found that the MamGFDC proteins are not essential for biomineralization, as the deletion of neither mamC, encoding the most abundant magnetosome protein, nor the entire mamGFDC operon abolished the formation of magnetite crystals. However, cells lacking mamGFDC produced crystals that were only 75% of the wild-type size and were less regular than wild-type crystals with respect to morphology and chain-like organization. The inhibition of crystal formation could not be eliminated by increased iron concentrations. The growth of mutant crystals apparently was not spatially constrained by the sizes of MM vesicles, as cells lacking mamGFDC formed vesicles with sizes and shapes nearly identical to those formed by wild-type cells. However, the formation of wild-type-size magnetite crystals could be gradually restored by in-trans complementation with one, two, and three genes of the mamGFDC operon, regardless of the combination, whereas the expression of all four genes resulted in crystals exceeding the wild-type size. Our data suggest that the MamGFDC proteins have partially redundant functions and, in a cumulative manner, control the growth of magnetite crystals by an as-yet-unknown mechanism.     

'Insulator bodies' are aggregates of proteins but not of insulators

Chromatin insulators are thought to restrict the action of enhancers and silencers. The best-known insulators in Drosophila require proteins such as Suppressor of Hairy wing (Su(Hw)) and Modifier of mdg4 (Mod(mdg4)) to be functional. The insulator-related proteins apparently colocalize as nuclear speckles in immunostained cells. It has been asserted that these speckles are 'insulator bodies' of many Su(Hw)-insulator DNA sites held together by associated proteins, including Mod(mdg4). As we show here using flies, larvae and S2 cells, a mutant Mod(mdg4) protein devoid of the Q-rich domain supports the function of Su(Hw)-dependent insulators and efficiently binds to correct insulator sites on the chromosome, but does not form or enter the Su(Hw)-marked nuclear speckles; conversely, the latter accumulate another (C-truncated) Mod(mdg4) mutant that cannot interact with Su(Hw) or with the genuine insulators. Hence, it is not the functional genomic insulators but rather aggregated proteins that make the so-called 'insulator bodies'.     

In vivo binding of wild-type and mutant human immunodeficiency virus type 1 Rev proteins: implications for function.

The Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1) is required for protein expression from the HIV-1 RNAs which contain a binding site for the Rev protein, termed the Rev-responsive element (RRE). This transactivator acts both at the level of splicing/transport of nuclear RNAs and at the level of translation of cytoplasmic RNAs. We used a monoclonal antibody specific for the HIV-1 Rev protein to immunoprecipitate cellular extracts from HIV-1-infected and -transfected cells. High levels of specific binding of wild-type Rev to the RRE-containing RNAs were found in cytoplasmic, but not nuclear, extracts from these cells. A Rev mutant which lacked both nuclear and cytoplasmic Rev function but retained RNA binding in vivo was generated. This binding was detectable with both nuclear and cytoplasmic extracts. These results verify the existence of direct binding of Rev to HIV-1 RNAs in vivo and conclusively prove that binding of Rev is not sufficient for nuclear or cytoplasmic Rev function. The results also support a direct role for Rev in the nuclear export and translation of HIV-1 RNAs.     

Escherichia coli MutS tetramerization domain structure reveals that stable dimers but not tetramers are essential for DNA mismatch repair in vivo

The Escherichia coli mispair-binding protein MutS forms dimers and tetramers in vitro, although the functional form in vivo is under debate. Here we demonstrate that the MutS tetramer is extended in solution using small angle x-ray scattering and the crystal structure of the C-terminal 34 amino acids of MutS containing the tetramer-forming domain fused to maltose-binding protein (MBP). Wild-type C-terminal MBP fusions formed tetramers and could bind MutS and MutS-MutL-DNA complexes. In contrast, D835R and R840E mutations predicted to disrupt tetrameric interactions only allowed dimerization of MBP. A chromosomal MutS truncation mutation eliminating the dimerization/tetramerization domain eliminated mismatch repair, whereas the tetramer-disrupting MutS D835R and R840E mutations only modestly affected MutS function. These results demonstrate that dimerization but not tetramerization of the MutS C terminus is essential for mismatch repair.     

The cysteine conserved among DNA cytosine methylases is required for methyl transfer, but not for specific DNA binding.

All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine. It has been proposed that this cysteine initiates catalysis by attacking the C6 of cytosine and thereby activating the normally inert C5 position. We show here that substitutions of this cysteine in the E. coli methylase M. EcoRII with either serine or tryptophan results in a complete loss of ability to transfer methyl groups to DNA. Interestingly, mutants with either serine or glycine substitution bind tightly to substrate DNA. These mutants resemble the wild-type enzyme in that their binding to substrate is not eliminated by the presence of non-specific DNA in the reaction, it is sensitive to methylation status of the substrate and is stimulated by an analog of the methyl donor. Hence the conserved cysteine is not essential for the specific stable binding of the enzyme to its substrate. However, substitution of the cysteine with the bulkier tryptophan does reduce DNA binding. We also report here a novel procedure for the synthesis of DNA containing 5-fluorocytosine. Further, we show that a DNA substrate for M. EcoRII in which the target cytosine is replaced by 5-fluorocytosine is a mechanism-based inhibitor of the enzyme and that it forms an irreversible complex with the enzyme. As expected, this modified substrate does not form irreversible complexes with the mutants.