scholarly journals DNA sequence context controls the binding and processivity of the T7 DNA primase

2018 ◽  
Author(s):  
Ariel Afek ◽  
Stefan Ilic ◽  
John Horton ◽  
David B. Lukatsky ◽  
Raluca Gordan ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequence ◽  
Functional Activity ◽  
Dna Recognition ◽  
Unique Combination ◽  
Binding Assays ◽  
Sequence Context ◽  
Sequence Composition ◽  
Complex Landscape ◽  
Biochemical Analyses

SUMMARYPrimases are key enzymes involved in DNA replication. They act on single-stranded DNA, and catalyze the synthesis of short RNA primers used by DNA polymerases. Here, we investigate the DNA-binding and activity of the bacteriophage T7 primase using a new workflow called High-Throughput Primase Profiling (HTPP). Using a unique combination of high-throughput binding assays and biochemical analyses, HTPP reveals a complex landscape of binding specificity and functional activity for the T7 primase, determined by sequences flanking the primase recognition site. We identified specific features, such as G/T-rich flanks, which increase primase-DNA binding up to 10-fold and, surprisingly, also increase the length of newly formed RNA (up to 3-fold). To our knowledge, variability in primer length has not been reported for this primase. We expect that applying HTPP to additional enzymes will reveal new insights into the effects of DNA sequence composition on the DNA recognition and functional activity of primases.

scholarly journals Rational design of a DNA sequence-specific modular protein tag by tuning the alkylation kinetics

2019 ◽  
Vol 10 (40) ◽  
pp. 9315-9325 ◽  
Author(s):  
Thang Minh Nguyen ◽  
Eiji Nakata ◽  
Zhengxiao Zhang ◽  
Masayuki Saimura ◽  
Huyen Dinh ◽  
...  
Keyword(s):  
Dna Binding ◽  
Kinetic Parameters ◽  
Dna Sequence ◽  
Rational Design ◽  
Design Principle ◽  
Dna Recognition ◽  
Protein Tag

A design principle for sequence-specific DNA modifiers driven by the specific DNA recognition was proposed based on the kinetic parameters for DNA binding and modification reactions.

scholarly journals DNA mismatches reveal widespread conformational penalties in protein-DNA recognition

2019 ◽  
Author(s):  
Ariel Afek ◽  
Honglue Shi ◽  
Atul Rangadurai ◽  
Harshit Sahay ◽  
Hashim M. Al-Hashimi ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Sequence ◽  
Dna Recognition ◽  
Energetic Cost ◽  
Base Pairs ◽  
Structural Distortions ◽  
Dna Mismatches ◽  
Specific Affinity ◽  
Dynamics Simulations ◽  
High Throughput Assay

ABSTRACTTranscription-factor (TF) proteins recognize specific genomic sequences, despite an overwhelming excess of non-specific DNA, to regulate complex gene expression programs1–3. While there have been significant advances in understanding how DNA sequence and shape contribute to recognition, some fundamental aspects of protein-DNA binding remain poorly understood2,3. Many DNA-binding proteins induce changes in the DNA structure outside the intrinsic B-DNA envelope. How the energetic cost associated with distorting DNA contributes to recognition has proven difficult to study and measure experimentally because the distorted DNA structures exist as low-abundance conformations in the naked B-DNA ensemble4–10. Here, we use a novel high-throughput assay called SaMBA (Saturation Mismatch-Binding Assay) to investigate the role of DNA conformational penalties in TF-DNA recognition. The approach introduces mismatched base-pairs (i.e. mispairs) within TF binding sites to pre-induce a variety of DNA structural distortions much larger than those induced by changes in Watson-Crick sequence. Strikingly, while most mismatches either weakened TF binding (~70%) or had negligible effects (~20%), approximately 10% of mismatches increased binding and at least one mismatch was found that increased the binding affinity for each of 21 examined TFs. Mismatches also converted sites from the non-specific affinity range into specific sites, and high-affinity sites into “super-sites” stronger than any known canonical binding site. These findings reveal a complex binding landscape that cannot be explained based on DNA sequence alone. Analysis of crystal structures together with NMR and molecular dynamics simulations revealed that many of the mismatches that increase binding induce distortions similar to those induced by TF binding, thus pre-paying some of the energetic cost to deform the DNA. Our work indicates that conformational penalties are a major determinant of protein-DNA recognition, and reveals mechanisms by which mismatches can recruit TFs and thus modulate replication and repair activities in the cell11,12.

scholarly journals Synthetic STARR-seq reveals how DNA shape and sequence modulate transcriptional output and noise

2018 ◽  
Author(s):  
Stefanie Schöne ◽  
Melissa Bothe ◽  
Edda Einfeldt ◽  
Marina Borschiwer ◽  
Philipp Benner ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Dna Sequence ◽  
Recognition Sequence ◽  
Similar Activity ◽  
Sequence Composition ◽  
Highly Active ◽  
Shape Characteristics ◽  
Dna Shape ◽  
Transcriptional Output

AbstractThe binding of transcription factors to short recognition sequences plays a pivotal role in controlling the expression of genes. The sequence and shape characteristics of binding sites influence DNA binding specificity and have also been implicated in modulating the activity of transcription factors downstream of binding. To quantitatively assess the transcriptional activity of dozens of thousands of designed synthetic sites in parallel, we developed a synthetic version of STARR-seq (synSTARR-seq). We used the approach to systematically analyze how variations in the recognition sequence of the glucocorticoid receptor (GR) affect transcriptional regulation. Our approach resulted in the identification of a novel highly active functional GR binding sequence and revealed that sequence variation both within and flanking GR’s core binding site can modulate GR activity without apparent changes in DNA binding affinity. Notably, we found that the sequence composition of variants with similar activity profiles was highly diverse. In contrast, groups of variants with similar activity profiles showed distinct DNA shape characteristics indicating that DNA shape may be a better predictor of activity than DNA sequence. Finally, using single cell experiments with individual enhancer variants, we obtained clues indicating that the architecture of the response element can independently tune expression mean and cell-to cell variability in gene expression (noise). Together, our studies establish synSTARR as a powerful method to systematically study how DNA sequence and shape modulate transcriptional output and noise.

scholarly journals The Small Subunit of M · AquI Is Responsible for Sequence-Specific DNA Recognition and Binding in the Absence of the Catalytic Domain

2003 ◽  
Vol 185 (4) ◽  
pp. 1284-1288 ◽  
Author(s):  
Hatice Pinarbasi ◽  
Ergun Pinarbasi ◽  
David P. Hornby
Keyword(s):  
Dna Binding ◽  
Dna Sequence ◽  
Dna Methyltransferase ◽  
Dna Recognition ◽  
Dna Methyltransferases ◽  
Binary Complex ◽  
Β Subunit ◽  
Α Subunit ◽  
Conserved Sequence ◽  
Covalent Complex

ABSTRACT AquI DNA methyltransferase (M · AquI) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5′-CCCGGG-3′. M · AquI is a heterodimer in which the polypeptide chain is separated at the junction between the two equivalent structural domains in the related enzyme M · HhaI. Recently, we reported the subcloning, overexpression, and purification of the subunits (α and β) of M · AquI separately. Here we describe the DNA binding properties of M · AquI. The results presented here indicate that the β subunit alone contains all of the information for sequence-specific DNA recognition and binding. The first step in the sequence-specific recognition of DNA by M · AquI involves the formation of binary complex with the target recognition domain in conjunction with conserved sequence motifs IX and X, found in all known C5 DNA methyltransferases, contained in the β subunit. The α subunit enhances the binding of the β subunit to DNA specifically and nonspecifically. It is likely that the addition of the α subunit to the β subunit stabilizes the conformation of the β subunit and thereby enhances its affinity for DNA indirectly. Addition of S-adenosyl-l-methionine and its analogues S-adenosyl-l-homocysteine and sinefungin enhances binding, but only in the presence of the α subunit. These compounds did not have any effect on DNA binding by the β subunit alone. Using a 30-mer oligodeoxynucleotide substrate containing 5-fluorodeoxycytidine (5-FdC), it was found that the β subunit alone did not form a covalent complex with its specific sequence in the absence or presence of S-adenosyl-l-methionine. However, the addition of the α subunit to the β subunit led to the formation of a covalent complex with specific DNA sequence containing 5-FdC.

scholarly journals Novel Transcriptional Regulons for Autotrophic Cycle Genes in Crenarchaeota

2015 ◽  
Vol 197 (14) ◽  
pp. 2383-2391 ◽  
Author(s):  
Semen A. Leyn ◽  
Irina A. Rodionova ◽  
Xiaoqing Li ◽  
Dmitry A. Rodionov
Keyword(s):  
Carbon Dioxide ◽  
Transcriptional Regulation ◽  
Comparative Genomics ◽  
Dna Binding ◽  
Transcriptional Control ◽  
Binding Assays ◽  
Promoter Regions ◽  
Content Type ◽  
Genome Context

ABSTRACTAutotrophic microorganisms are able to utilize carbon dioxide as their only carbon source, or, alternatively, many of them can grow heterotrophically on organics. Different variants of autotrophic pathways have been identified in various lineages of the phylumCrenarchaeota. Aerobic members of the orderSulfolobalesutilize the hydroxypropionate-hydroxybutyrate cycle (HHC) to fix inorganic carbon, whereas anaerobicThermoprotealesuse the dicarboxylate-hydroxybutyrate cycle (DHC). Knowledge of transcriptional regulation of autotrophic pathways inArchaeais limited. We applied a comparative genomics approach to predict novel autotrophic regulons in theCrenarchaeota. We report identification of two novel DNA motifs associated with the autotrophic pathway genes in theSulfolobales(HHC box) andThermoproteales(DHC box). Based on genome context evidence, the HHC box regulon was attributed to a novel transcription factor from the TrmB family named HhcR. Orthologs of HhcR are present in allSulfolobalesgenomes but were not found in other lineages. A predicted HHC box regulatory motif was confirmed byin vitrobinding assays with the recombinant HhcR protein fromMetallosphaera yellowstonensis. For the DHC box regulon, we assigned a different potential regulator, named DhcR, which is restricted to the orderThermoproteales. DhcR inThermoproteus neutrophilus(Tneu_0751) was previously identified as a DNA-binding protein with high affinity for the promoter regions of two autotrophic operons. The global HhcR and DhcR regulons reconstructed by comparative genomics were reconciled with available omics data inMetallosphaeraandThermoproteusspp. The identified regulons constitute two novel mechanisms for transcriptional control of autotrophic pathways in theCrenarchaeota.IMPORTANCELittle is known about transcriptional regulation of carbon dioxide fixation pathways inArchaea. We previously applied the comparative genomics approach for reconstruction of DtxR family regulons in diverse lineages ofArchaea. Here, we utilize similar computational approaches to identify novel regulatory motifs for genes that are autotrophically induced in microorganisms from two lineages ofCrenarchaeotaand to reconstruct the respective regulons. The predicted novel regulons in archaeal genomes control the majority of autotrophic pathway genes and also other carbon and energy metabolism genes. The HhcR regulon was experimentally validated by DNA-binding assays inMetallosphaeraspp. Novel regulons described for the first time in this work provide a basis for understanding the mechanisms of transcriptional regulation of autotrophic pathways inArchaea.

scholarly journals Androgen and glucocorticoid receptor direct distinct transcriptional programs by receptor-specific and shared DNA binding sites

2021 ◽  
Vol 49 (7) ◽  
pp. 3856-3875
Author(s):  
Marina Kulik ◽  
Melissa Bothe ◽  
Gözde Kibar ◽  
Alisa Fuchs ◽  
Stefanie Schöne ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Transcription Factor ◽  
Dna Binding ◽  
Receptor Binding ◽  
Binding Sites ◽  
Specific Gene ◽  
Functional Diversification ◽  
Enhancer Activity ◽  
Sequence Composition

Abstract The glucocorticoid (GR) and androgen (AR) receptors execute unique functions in vivo, yet have nearly identical DNA binding specificities. To identify mechanisms that facilitate functional diversification among these transcription factor paralogs, we studied them in an equivalent cellular context. Analysis of chromatin and sequence suggest that divergent binding, and corresponding gene regulation, are driven by different abilities of AR and GR to interact with relatively inaccessible chromatin. Divergent genomic binding patterns can also be the result of subtle differences in DNA binding preference between AR and GR. Furthermore, the sequence composition of large regions (>10 kb) surrounding selectively occupied binding sites differs significantly, indicating a role for the sequence environment in guiding AR and GR to distinct binding sites. The comparison of binding sites that are shared shows that the specificity paradox can also be resolved by differences in the events that occur downstream of receptor binding. Specifically, shared binding sites display receptor-specific enhancer activity, cofactor recruitment and changes in histone modifications. Genomic deletion of shared binding sites demonstrates their contribution to directing receptor-specific gene regulation. Together, these data suggest that differences in genomic occupancy as well as divergence in the events that occur downstream of receptor binding direct functional diversification among transcription factor paralogs.

scholarly journals Influence of supercoiling and sequence context on operator DNA binding with lac repressor.

1987 ◽  
Vol 262 (30) ◽  
pp. 14592-14599 ◽  
Author(s):  
P A Whitson ◽  
W T Hsieh ◽  
R D Wells ◽  
K S Matthews
Keyword(s):  
Dna Binding ◽  
Lac Repressor ◽  
Sequence Context ◽  
Operator Dna

scholarly journals Genome-Wide Binding Analyses of HOXB1 Revealed a Novel DNA Binding Motif Associated with Gene Repression

2021 ◽  
Vol 9 (1) ◽  
pp. 6
Author(s):  
Narendra Pratap Singh ◽  
Bony De Kumar ◽  
Ariel Paulson ◽  
Mark E. Parrish ◽  
Carrie Scott ◽  
...  
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Binding Assays ◽  
Histone Marks ◽  
Genome Wide ◽  
Hox Cofactors

Knowledge of the diverse DNA binding specificities of transcription factors is important for understanding their specific regulatory functions in animal development and evolution. We have examined the genome-wide binding properties of the mouse HOXB1 protein in embryonic stem cells differentiated into neural fates. Unexpectedly, only a small number of HOXB1 bound regions (7%) correlate with binding of the known HOX cofactors PBX and MEIS. In contrast, 22% of the HOXB1 binding peaks display co-occupancy with the transcriptional repressor REST. Analyses revealed that co-binding of HOXB1 with PBX correlates with active histone marks and high levels of expression, while co-occupancy with REST correlates with repressive histone marks and repression of the target genes. Analysis of HOXB1 bound regions uncovered enrichment of a novel 15 base pair HOXB1 binding motif HB1RE (HOXB1 response element). In vitro template binding assays showed that HOXB1, PBX1, and MEIS can bind to this motif. In vivo, this motif is sufficient for direct expression of a reporter gene and over-expression of HOXB1 selectively represses this activity. Our analyses suggest that HOXB1 has evolved an association with REST in gene regulation and the novel HB1RE motif contributes to HOXB1 function in part through a repressive role in gene expression.

scholarly journals DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants

2008 ◽  
Vol 283 (51) ◽  
pp. 35569-35578 ◽  
Author(s):  
Yelena Margolin ◽  
Vladimir Shafirovich ◽  
Nicholas E. Geacintov ◽  
Michael S. DeMott ◽  
Peter C. Dedon
Keyword(s):  
Dna Sequence ◽  
Hydroxyl Radicals ◽  
Sequence Context ◽  
Guanine Oxidation
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