Determinants in the sequence specific binding of two plant transcription factors, CBF1 and NtERF2, to the DRE and GCC motifs

Arabidopsis ERF proteins such as DREB1, DREB2, and CBF1 bind to the dehydration-responsive element (DRE), which has the sequence TACCGACAT. Mutation analyses reveal that a central 5 bp CCGAC core of the DRE is the minimal sequence motif (designated as the DRE motif in this paper), to which the ERF domain fragment of CBF1 (CBF1-F) binds specifically with a binding K(d) at the nanomolar level. In contrast, the ERF domain fragment of the tobacco ERF2 (NtERF2-F) does not interact with the DRE motif, but restrictedly recognizes the sequence containing a minimal 6 bp GCCGCC motif (designated as the GCC motif in this paper). However, CBF1-F binds to the GCC motif with a binding activity similar to its binding activity for the DRE motif. These in vitro binding variations were further demonstrated through reporter cotransformation assays, suggesting that the DRE and GCC motifs are two similar sequence motifs sharing a common core region of CCGNC with a discriminating guanine base at the 5'-end of the GCC motif. Binding analyses with the mutated ERF domain show that such a unique binding of NtERF2-F to the GCC motif can be altered by the substitution of A14 with valine in beta-strand 2 of its ERF domain, the mutant NtERF2-F, ERFav, acquiring a binding to the DRE motif with a K(d) comparable to that for CBF1-F binding to the DRE motif. This demonstrates that A14 is an important determinant of the NtERF2-F binding specificity. A possible mechanism of the binding specificity determination is discussed.     

Candidate cis-elements for human renin gene expression in the promoter region

The regulation of renin gene expression, the rate‐limiting enzyme of the system, is thought to be fundamental to the total system. Previously, we mapped six putative cis‐elements in the promoter region of the human renin gene with nuclear proteins from human chorionic cells and human renal cortex by DNase I protection assay (footprint A–F). Each footprint contains Ets motif like site (A), HOXñPBX recognition sequence (B), unknown sequence as DNA binding consensus (C), CRE (D), COUP‐TFII (ARP‐1) motif like site (E), and AGE3 like site (F). Footprint D has been characterized by means of functional studies as the genuine human renin gene CRE interacting with CREB in cooperation with the site of footprint B. To obtain further clues to the specific expression in the promoter region, these putative cis‐elements were conducted to a consensus‐specific binding assay to compare renin‐producing and non‐renin‐producing cells by EMSA and electromobility super‐shift assay. Different sequence‐specific DNA/protein binding was obtained among the different cell lines with footprint B site, with COUP‐TFII (ARP‐1) motif like site and possibly with footprint F site. The results implicate these putative cis‐elements and each corresponding trans‐factor in the specific expression of the human renin gene in the promoter region. Further functional characterization of these elements would provide important data for a better understanding of human renin gene expression. © 2004 Wiley‐Liss, Inc.     

A Conserved Sequence Extending Motif III of the Motor Domain in the Snf2-Family DNA Translocase Rad54 Is Critical for ATPase Activity

Rad54 is a dsDNA-dependent ATPase that translocates on duplex DNA. Its ATPase function is essential for homologous recombination, a pathway critical for meiotic chromosome segregation, repair of complex DNA damage, and recovery of stalled or broken replication forks. In recombination, Rad54 cooperates with Rad51 protein and is required to dissociate Rad51 from heteroduplex DNA to allow access by DNA polymerases for recombination-associated DNA synthesis. Sequence analysis revealed that Rad54 contains a perfect match to the consensus PIP box sequence, a widely spread PCNA interaction motif. Indeed, Rad54 interacts directly with PCNA, but this interaction is not mediated by the Rad54 PIP box-like sequence. This sequence is located as an extension of motif III of the Rad54 motor domain and is essential for full Rad54 ATPase activity. Mutations in this motif render Rad54 non-functional in vivo and severely compromise its activities in vitro. Further analysis demonstrated that such mutations affect dsDNA binding, consistent with the location of this sequence motif on the surface of the cleft formed by two RecA-like domains, which likely forms the dsDNA binding site of Rad54. Our study identified a novel sequence motif critical for Rad54 function and showed that even perfect matches to the PIP box consensus may not necessarily identify PCNA interaction sites.     

Nucleoporation of dendritic cells: efficient gene transfer by electroporation into human monocyte-derived dendritic cells

Four new members of the ERF (ethylene-response factor) family of plant-specific DNA-binding (GCC box) factors were isolated from tomato fruit (LeERF1–4). Phylogenetic analysis indicated that LeERF2 belongs to a new ERF class, characterized by a conserved N-terminal signature sequence. Expression patterns and cis/trans binding affinities differed between the LeERFs. Combining experimental data and modeled three-dimensional analysis, it was shown that binding affinity of the LeERFs was affected by both the variation of nucleotides surrounding the DNA cis-element sequence and the nature of critical amino acid residues within the ERF domain.     

Conformational diversity of single‐stranded DNA from bacterial repetitive extragenic palindromes: Implications for the DNA recognition elements of transposases

Repetitive extragenic palindrome (REP)—associated tyrosine transposase enzymes (RAYTs) bind REP DNA domains and catalyze their cleavage. Genomic sequence analyses identify potential noncoding REP sequences associated with RAYT‐encoding genes. To probe the conformational space of potential RAYT DNA binding domains, we report here spectroscopic and calorimetric measurements that detect and partially characterize the solution conformational heterogeneity of REP oligonucleotides from six bacterial species. Our data reveal most of these REP oligonucleotides adopt multiple conformations, suggesting that RAYTs confront a landscape of potential DNA substrates in dynamic equilibrium that could be selected, enriched, and/or induced via differential binding. Thus, the transposase‐bound DNA motif may not be the predominant conformation of the isolated REP domain. Intriguingly, for several REPs, the circular dichroism spectra suggest guanine tetraplexes as potential alternative or additional RAYT recognition elements, an observation consistent with these REP domains being highly nonrandom, with tetraplex‐favoring 5′‐G and 3′‐C‐rich segments. In fact, the conformational heterogeneity of REP domains detected and reported here, including the formation of noncanonical DNA secondary structures, may reflect a general feature required for recognition by RAYT transposases. Based on our biophysical data, we propose guanine tetraplexes as an additional DNA recognition element for binding by RAYT transposase enzymes. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 585–596, 2015.     

Genome-wide analysis of transcription factor E2F1 mutant proteins reveals that N- and C-terminal protein interaction domains do not participate in targeting E2F1 to the human genome

Previous studies of E2F family members have suggested that protein-protein interactions may be the mechanism by which E2F proteins are recruited to specific genomic regions. We have addressed this hypothesis on a genome-wide scale using ChIP-seq analysis of MCF7 cell lines that express tagged wild type and mutant E2F1 proteins. First, we performed ChIP-seq for tagged WT E2F1. Then, we analyzed E2F1 proteins that lacked the N-terminal SP1 and cyclin A binding domains, the C-terminal transactivation and pocket protein binding domains, and the internal marked box domain. Surprisingly, we found that the ChIP-seq patterns of the mutant proteins were identical to that of WT E2F1. However, mutation of the DNA binding domain abrogated all E2F1 binding to the genome. These results suggested that the interaction between the E2F1 DNA binding domain and a consensus motif may be the primary determinant of E2F1 recruitment. To address this possibility, we analyzed the in vivo binding sites for the in vitro-derived consensus E2F1 motif (TTTSSCGC) and also performed de novo motif analysis. We found that only 12% of the ChIP-seq peaks contained the TTTSSCGC motif. De novo motif analysis indicated that most of the in vivo sites lacked the 5' half of the in vitro-derived consensus, having instead the in vivo consensus of CGCGC. In summary, our findings do not provide support for the model that protein-protein interactions are involved in recruiting E2F1 to the genome, but rather suggest that recognition of a motif found at most human promoters is the critical determinant.     

Structure of the Rad50 DNA double‐strand break repair protein in complex with DNA

The Mre11–Rad50 nuclease–ATPase is an evolutionarily conserved multifunctional DNA double‐strand break (DSB) repair factor. Mre11–Rad50's mechanism in the processing, tethering, and signaling of DSBs is unclear, in part because we lack a structural framework for its interaction with DNA in different functional states. We determined the crystal structure of Thermotoga maritima Rad50^NBD (nucleotide‐binding domain) in complex with Mre11^HLH (helix‐loop‐helix domain), AMPPNP, and double‐stranded DNA. DNA binds between both coiled‐coil domains of the Rad50 dimer with main interactions to a strand‐loop‐helix motif on the NBD. Our analysis suggests that this motif on Rad50 does not directly recognize DNA ends and binds internal sites on DNA. Functional studies reveal that DNA binding to Rad50 is not critical for DNA double‐strand break repair but is important for telomere maintenance. In summary, we provide a structural framework for DNA binding to Rad50 in the ATP‐bound state.     

Mutational analysis of the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase

On the basis of amino acid sequence alignments and structural data of related enzymes, we have performed a mutational analysis of 14 amino acid residues in the catalytic domain of the murine Dnmt3a DNA-(cytosine C5)-methyltransferase. The target residues are located within the ten conserved amino acid sequence motifs characteristic for cytosine-C5 methyltransferases and in the putative DNA recognition domain of the enzyme (TRD). Mutant proteins were purified and tested for their catalytic properties and their abilities to bind DNA and AdoMet. We prepared a structural model of Dnmt3a to interpret our results. We demonstrate that Phe50 (motif I) and Glu74 (motif II) are important for AdoMet binding and catalysis. D96A (motif III) showed reduced AdoMet binding but increased activity under conditions of saturation with S-adenosyl-L-methionine (AdoMet), indicating that the contact of Asp96 to AdoMet is not required for catalysis. R130A (following motif IV), R241A and R246A (in the TRD), R292A, and R297A (both located in front of motif X) showed reduced DNA binding. R130A displayed a strong reduction in catalytic activity and a complete change in flanking sequence preferences, indicating that Arg130 has an important role in the DNA interaction of Dnmt3a. R292A also displayed reduced activity and changes in the flanking sequence preferences, indicating a potential role in DNA contacts farther away from the CG target site. N167A (motif VI) and R202A (motif VIII) have normal AdoMet and DNA binding but reduced catalytic activity. While Asn167 might contribute to the positioning of residues from motif VI, according to structural data Arg202 has a role in catalysis of cytosine-C5 methyltransferases. The R295A variant was catalytically inactive most likely because of destabilization of the hinge sub-domain of the protein.