Molecular architecture of the DNA-binding sites of the P-loop ATPases MipZ and ParA from Caulobacter crescentus

Abstract The spatiotemporal regulation of chromosome segregation and cell division in Caulobacter crescentus is mediated by two different P-loop ATPases, ParA and MipZ. Both of these proteins form dynamic concentration gradients that control the positioning of regulatory targets within the cell. Their proper localization depends on their nucleotide-dependent cycling between a monomeric and a dimeric state and on the ability of the dimeric species to associate with the nucleoid. In this study, we use a combination of genetic screening, biochemical analysis and hydrogen/deuterium exchange mass spectrometry to comprehensively map the residues mediating the interactions of MipZ and ParA with DNA. We show that MipZ has non-specific DNA-binding activity that relies on an array of positively charged and hydrophobic residues lining both sides of the dimer interface. Extending our analysis to ParA, we find that the MipZ and ParA DNA-binding sites differ markedly in composition, although their relative positions on the dimer surface and their mode of DNA binding are conserved. In line with previous experimental work, bioinformatic analysis suggests that the same principles may apply to other members of the P-loop ATPase family. P-loop ATPases thus share common mechanistic features, although their functions have diverged considerably during the course of evolution.

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Molecular architecture of the DNA‐binding sites of the P‐loop ATPases MipZ and ParA from Caulobacter crescentus

10.1101/766287 ◽

2019 ◽

Author(s):

Yacine Refes ◽

Binbin He ◽

Laura Corrales-Guerrero ◽

Wieland Steinchen ◽

Gaël Panis ◽

...

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Electrostatic Interactions ◽

Caulobacter Crescentus ◽

Bioinformatic Analysis ◽

Proper Function ◽

Molecular Architecture ◽

Independent Manner ◽

Dna Binding Sites

ABSTRACTTwo related P-loop ATPases, ParA and MipZ, mediate the spatiotemporal regulation of chromosome segregation and cell division in Caulobacter crescentus. Both of these proteins share the ability to form dynamic concentration gradients that control the positioning of regulatory targets within the cell. Their proper localization relies on their nucleotide-dependent cycling between a monomeric and a dimeric state, driven by interaction with the chromosome partitioning protein ParB, and on the ability of the dimeric species to associate non-specifically with the nucleoid. In this study, we use a combination of genetic screening, biochemical analysis and hydrogen/deuterium exchange mass spectrometry to identify the residues mediating the interaction of MipZ with DNA. Our results show that the DNA-binding activity of MipZ relies on a series of positively charged and hydrophobic residues lining both sides of the dimer interface. MipZ thus appears to associate with DNA in a sequence-independent manner through electrostatic interactions with the DNA phosphate backbone. In support of this hypothesis, chromatin immunoprecipitation analyses did not reveal any specific target sites in vivo. When extending our analysis to ParA, we found that the architectures of the MipZ and ParA DNA-binding sites are markedly different, although their relative positions on the dimer surface and their mode of DNA binding are conserved. Importantly, bioinformatic analysis suggests that the same principles apply to other members of the P-loop ATPase family. ParA-like ATPases thus share common mechanistic features, although their modes of action have diverged considerably during the course of evolution.SIGNIFICANCEParA-like P-loop ATPases are involved in a variety of cellular processes in bacteria, including chromosome and plasmid segregation, chemoreceptor and carboxysome positioning, and division site placement. Many members of this large protein family depend on the ability to bind non-specific DNA for proper function. Although previous studies have yielded insights in the DNA-binding properties of some ParA-like ATPases, a comprehensive view of the underlying mechanisms is still lacking. Here, we combine state-of-the-art cell biological, biochemical and biophysical approaches to localize the DNA-binding regions of the ParA-like ATPases MipZ and ParA from Caulobacter crescentus. We show that the two proteins use the same interface and mode of action to associate with DNA, suggesting that the mechanistic basis of DNA binding may be conserved in the ParA-like ATPase family.

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Androgen-dependent and DNA binding-independent association of androgen receptor with chromatic regions coding androgen-induced non-coding RNAs

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab135 ◽

2021 ◽

Author(s):

Takahiro Sawada ◽

Koichi Nishimura ◽

Jinichi Mori ◽

Yoshiaki Kanemoto ◽

Alexander Kouzmenko ◽

...

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Specific Antigen ◽

Binding Activity ◽

Lncap Cells ◽

Coding Regions ◽

Dna Binding Activity ◽

Genome Wide ◽

Non Coding Rnas ◽

Independent Association

Abstract Androgen induces the binding of its receptor (AR) to androgen-responsive elements (AREs), while genome-wide studies showed that most androgen-induced AR binding sites on chromatin were unrelated to AREs. Enhancer RNAs (eRNAs), a class of non-coding RNAs(ncRNAs), are transcribed from super-enhancers (SEs), and trigger the formation of large ribonucleoprotein (RNP) condensates of transcription factors. By in silico search, an SE is found to be located on the locus of KLK3 that encodes prostate specific antigen (PSA). On the KLK3 SE, androgen-induced expression of ncRNAs was detected and designated as KLK3eRNAs in LNCaP cells, and androgen-induced association of AR and FOXA1 on the KLK3eRNA coding regions was detected. Such androgen-induced association of an AR mutant lacking DNA binding activity on the KLK3eRNA coding regions was undetectable on an exogenous ARE. Thus, the present findings suggest a molecular basis of androgen-induced association of AR with chromatin on ARE-unrelated sequences.

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DNA-binding properties of the Drosophila melanogaster zeste gene product.

Molecular and Cellular Biology ◽

10.1128/mcb.8.2.615 ◽

1988 ◽

Vol 8 (2) ◽

pp. 615-623 ◽

Cited By ~ 11

Author(s):

A Mansukhani ◽

A Crickmore ◽

P W Sherwood ◽

M L Goldberg

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Binding Sites ◽

Fusion Proteins ◽

Genetic Interaction ◽

Binding Activity ◽

Transcriptional Unit ◽

Dna Binding Activity ◽

Zinc Finger Motifs ◽

White Locus

The ability of the zeste moiety of beta-galactosidase-zeste fusion proteins synthesized in Escherichia coli to bind specific DNA sequences was examined. Such fusion proteins recognize a region of the white locus upstream of the start of transcription; this region has previously been shown to be required for genetic interaction between the zeste and white loci. Another strong binding site was localized to a region between 50 and 205 nucleotides before the start of the Ubx transcriptional unit; expression of the bithorax complex is also known to be influenced by the zeste locus. Weaker binding sites were also seen in the vicinity of the bxd and Sgs-4 genes, but it is currently unclear whether these binding sites play a role in transvection effects. The DNA-binding activity of the zeste protein is restricted to a domain of approximately 90 amino acids near the N terminus. This domain does not appear to contain homeobox or zinc finger motifs found in other DNA-binding proteins. The DNA-binding domain is not disrupted by any currently characterized zeste mutations.

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Efficient transcription of the glycolytic gene ADH1 and three translational component genes requires the GCR1 product, which can act through TUF/GRF/RAP binding sites

Molecular and Cellular Biology ◽

10.1128/mcb.10.2.859-862.1990 ◽

1990 ◽

Vol 10 (2) ◽

pp. 859-862

Author(s):

G M Santangelo ◽

J Tornow

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Dna Binding ◽

Binding Sites ◽

Binding Activity ◽

Heterologous Gene ◽

Dna Binding Activity ◽

Glycolytic Gene

Glycolytic gene expression in Saccharomyces cerevisiae is thought to be activated by the GCR and TUF proteins. We tested the hypothesis that GCR function is mediated by TUF/GRF/RAP binding sites (UASRPG elements). We found that UASRPG-dependent activation of a heterologous gene and transcription of ADH1, TEF1, TEF2, and RP59 were sensitive to GCR1 disruption. GCR is not required for TUF/GRF/RAP expression or in vitro DNA-binding activity.

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One exon of the human LSF gene includes conserved regions involved in novel DNA-binding and dimerization motifs

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5076-5087.1994 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5076-5087

Author(s):

M K Shirra ◽

Q Zhu ◽

H C Huang ◽

D Pallas ◽

U Hansen

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Simian Virus 40 ◽

Simian Virus ◽

Binding Activity ◽

Protein Protein Interaction ◽

Dna Binding Activity ◽

Dna Binding Sites ◽

Alternative Mrna Splicing ◽

Alternatively Spliced

The transcription factor LSF, identified as a HeLa protein that binds the simian virus 40 late promoter, recognizes direct repeats with a center-to-center spacing of 10 bp. The characterization of two human cDNAs, representing alternatively spliced mRNAs, provides insight into the unusual DNA-binding and oligomerization properties of LSF. The sequence of the full-length LSF is identical to that of the transcription factors alpha CP2 and LBP-1c and has similarity to the Drosophila transcription factor Elf-1/NTF-1. Using an epitope-counting method, we show that LSF binds DNA as a homodimer. LSF-ID, which is identical to LBP-1d, contains an in-frame internal deletion of 51 amino acids resulting from alternative mRNA splicing. Unlike LSF, LSF-ID did not bind LSF DNA-binding sites. Furthermore, LSF-ID did not affect the binding of LSF to DNA, suggesting that the two proteins do not interact. Of three short regions with a high degree of homology between LSF and Elf-1/NTF-1, LSF-ID lacks two, which are predicted to form beta-strands. Double amino acid substitutions in each of these regions eliminated specific DNA-binding activity, similarly to the LSF-ID deletion. The dimerization potential of these mutants was measured both by the ability to inhibit the binding of LSF to DNA and by direct protein-protein interaction studies. Mutations in one homology region, but not the other, functionally eliminated dimerization.

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The interaction of proteins encoded by Drosophila homeotic and segmentation genes with specific DNA sequences

Development ◽

10.1242/dev.104.supplement.75 ◽

1988 ◽

Vol 104 (Supplement) ◽

pp. 75-83 ◽

Cited By ~ 2

Author(s):

Allen Laughon ◽

William Howell ◽

Matthew P. Scott

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Binding Sites ◽

Binding Activity ◽

Regulatory Function ◽

Temperature Sensitive ◽

Structural Domain ◽

Dna Binding Activity ◽

Dna Restriction Fragments

The ANT-C gene cluster is part of a network of genes that govern pattern formation in the development of Drosophila. The ANT-C genes encode proteins that contain a conserved 60 amino acid sequence, the homeodomain. Here we show that the homeodomains encoded by two of the ANT-C loci confer sequencespecific DNA-binding activity. The DNA sequence specificities of the Dfd and ftz homeodomains appear to overlap completely in vitro, indicating that differences in regulatory specificity among ANT-C and BX-C proteins (assuming that differences exist) must be a consequence of the nonconserved protein sequences found outside of the homeodomains. Deletions that remove sequences from either end of the ftz homeodomain abolish DNA-binding activity, consistent with the commonly held assumption that the homeodomain is a structural domain. The relevance of in vitro DNA-binding experiments to the regulatory function of ftz is supported by our finding that a temperature-sensitive ftz mutation that causes a pairwise fusion of embryonic segments also reduces the affinity of the ftz homeodomain for DNA. Restriction fragments containing ftz homeodomain binding sites were identified within a 90 kb stretch of DNA extending the Antp P1 and P2 promoters. Binding sites appear to be clustered near the P1 promoter but also occur near P2 and in the region between the two. The task remains of determining which of these sequences mediate regulation of Antp by ftz or by other genes that encode closely related homeodomains.

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Protein-DNA interactions at the major and minor promoters of the divergently transcribed dhfr and rep3 genes during the Chinese hamster ovary cell cycle.

Molecular and Cellular Biology ◽

10.1128/mcb.16.2.634 ◽

1996 ◽

Vol 16 (2) ◽

pp. 634-647 ◽

Cited By ~ 32

Author(s):

J Wells ◽

P Held ◽

S Illenye ◽

N H Heintz

Keyword(s):

Phase Transition ◽

Cell Cycle ◽

Dna Binding ◽

Binding Sites ◽

Binding Activity ◽

Dnase I ◽

S Phase ◽

Ovary Cell ◽

Dna Binding Activity ◽

Genomic Footprinting

In mammals, two TATA-less bidirectional promoters regulate expression of the divergently transcribed dihydrofolate reductase (dhfr) and rep3 genes. In CHOC 400 cells, dhfr mRNA levels increase about fourfold during the G1-to-S phase transition of the cell cycle, whereas the levels of rep3 transcripts vary less than twofold during this time. To assess the role of DNA-binding proteins in transcriptional regulation of the dhfr and rep3 genes, the major and minor dhfr-rep3 promoter regions were analyzed by high-resolution genomic footprinting during the cell cycle. At the major dhfr promoter, prominent DNase I footprints over four upstream Sp1 binding sites did not vary throughout G1 and entry into the S phase. Genomic footprinting revealed that a protein is constitutively bound to the overlapping E2F sites throughout the G1-to-S phase transition, an interaction that is most evident on the transcribed template strand. On the nontranscribed strand, multiple changes in the DNase I cleavage pattern are observed during transit through G1 and entry into the S phase. By using gel mobility shift assays and a series of sequence-specific probes, two different species of E2F were shown to interact with the dhfr promoter during the cell cycle. The DNA binding activity of one E2F species, which preferentially recognizes the sequence TTTGGCGC, did not vary significantly during the cell cycle. The DNA binding activity of the second E2F species, which preferentially recognizes the sequence TTTCGCGC, increased during the G1-to-S phase transition. Together, these results indicate that Sp1 and the species of E2F that binds TTTGGCGC participate in the formation of a basal transcription complex, while the species of E2F that binds TTTCGCGC regulates dhfr gene expression during the G1-to-S phase transition. At the minor promoter, DNase I footprints at a consensus c-Myc binding site and three Sp1 binding sites showed little variation during the G1-to-S phase transition. In addition to protein binding at sequences known to be involved in the regulation of transcription, genomic footprinting of the entire promoter region also showed that a protein factor is constitutively bound to the first intron of the rep3 gene.

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Functional Characterization of a Testis-Specific DNA Binding Activity at the H19/Igf2 Imprinting Control Region

Molecular and Cellular Biology ◽

10.1128/mcb.23.22.8345-8351.2003 ◽

2003 ◽

Vol 23 (22) ◽

pp. 8345-8351 ◽

Cited By ~ 15

Author(s):

Aaron B. Bowman ◽

John M. Levorse ◽

Robert S. Ingram ◽

Shirley M. Tilghman

Keyword(s):

Dna Methylation ◽

Dna Binding ◽

Control Region ◽

Binding Sites ◽

Germ Line ◽

Binding Activity ◽

Wild Type ◽

Dna Binding Activity ◽

Epigenetic State ◽

Imprinting Control Region

ABSTRACT The DNA methylation state of the H19/Igf2 imprinting control region (ICR) is differentially set during gametogenesis. To identify factors responsible for the paternally specific DNA methylation of the ICR, germ line and somatic extracts were screened for proteins that bind to the ICR in a germ line-specific manner. A specific DNA binding activity that was restricted to the male germ line and enriched in neonatal testis was identified. Its three binding sites within the ICR are very similar to the consensus sequence for nuclear receptor extended half sites. To determine if these binding sites are required for establishment of the paternal epigenetic state, a mouse strain in which the three sites were mutated was generated. The mutated ICR was able to establish a male-specific epigenetic state in sperm that was indistinguishable from that established by the wild-type ICR, indicating that these sequences are either redundant or have no function. An analysis of the methylated state of the mutant ICR in the soma revealed no differences from the wild-type ICR but did uncover in both mutant and wild-type chromosomes a significant relaxation in the stringency of the methylated state of the paternal allele and the unmethylated state of the maternal allele in neonatal and adult tissues.

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