Increased DNA Flexibility Research Articles

Enhanced nucleosome assembly at CpG sites containing an extended 5-methylcytosine analogue.

Methylation of cytosine to 5-methylcytosine (mC) at CpG sites is a prevalent reversible epigenetic mark in vertebrates established by DNA methyltransferases (MTases); the attached methyl groups can alter local structure of DNA and chromatin as well as binding of dedicated proteins. Nucleosome assembly on methylated DNA has been studied extensively, however little is known how the chromatin structure is affected by larger chemical variations in the major groove of DNA. Here, we studied the nucleosome formation in vitro on DNA containing an extended 5mC analog, 5-(6-azidohex-2-ynyl)cytosine (ahyC) installed at biological relevant CpG sites. We found that multiple ahyC residues on 80-Widom and Hsp70 promoter DNA fragments proved compatible with nucleosome assembly. Moreover, unlike mC, ahyC increases the affinity of histones to the DNA, partially altering nucleosome positioning, stability, and the action of chromatin remodelers. Based on molecular dynamics calculations, we suggest that these new features are due to increased DNA flexibility at ahyC-modified sites. Our findings provide new insights into the biophysical behavior of modified DNA and open new ways for directed design of synthetic nucleosomes.

Single-molecule micromanipulation studies of methylated DNA

Cytosine methylated at the five-carbon position is the most widely studied reversible DNA modification. Prior findings indicate that methylation can alter mechanical properties. However, those findings were qualitative and sometimes contradictory, leaving many aspects unclear. By applying single-molecule magnetic force spectroscopy techniques allowing for direct manipulation and dynamic observation of DNA mechanics and mechanically driven strand separation, we investigated how CpG and non-CpG cytosine methylation affects DNA micromechanical properties. We quantitatively characterized DNA stiffness using persistence length measurements from force-extension curves in the nanoscale length regime and demonstrated that cytosine methylation results in longer contour length and increased DNA flexibility (i.e., decreased persistence length). In addition, we observed the preferential formation of plectonemes over unwound single-stranded “bubbles” of DNA under physiologically relevant stretching forces and supercoiling densities. The flexibility and high structural stability of methylated DNA is likely to have significant consequences on the recruitment of proteins recognizing cytosine methylation and DNA packaging.

CRISPR-Cas12a Nucleases Bind Flexible DNA Duplexes Without RNA-DNA Complementarity

Cas12a (also known as “Cpf1”) is a class 2 type V CRISPR-associated nuclease that can cleave double-stranded DNA at specific sites. The Cas12a effector enzyme comprises of a single protein and a CRISPR-encoded small RNA (crRNA), and has been used for genome editing and manipulation. Work reported here examined in vitro interactions between the Cas12a effector enzyme and DNA duplexes with varying states of base-pairing between the two strands. The data revealed that in the absence of complementarity between the crRNA guide and the DNA target-strand, Cas12a binds duplexes with unpaired segments. These off-target duplexes were bound at the Cas12a site responsible for RNA-guided double-stranded DNA binding, but were not cleaved due to the lack of RNA/DNA hybrid formation. Such promiscuous binding was attributed to increased DNA flexibility induced by the unpaired segment present next to the protospacer-adjacent-motif. The results suggest that target discrimination of Cas12a can be influenced by flexibility of the DNA. As such, in addition to the linear sequence, flexibility and other physical properties of the DNA should be considered in Cas12a-based genome engineering applications.

Deciphering the Gene Regulatory Landscape Encoded in DNA Biophysical Features.

SummaryGene regulation in higher organisms involves a sophisticated interplay between genetic and epigenetic mechanisms. Despite advances, the logic in selective usage of certain genomic regions as regulatory elements remains unclear. Here we show that the inherent biophysical properties of the DNA encode epigenetic state and the underlying regulatory potential. We find that the propeller twist (ProT) level is indicative of genomic location of the regulatory elements, their strength, the affinity landscape of transcription factors, and distribution in the nuclear 3D space. We experimentally show that ProT levels confer increased DNA flexibility and surface accessibility, and thus potentially primes usage of high ProT regions as regulatory elements. ProT levels also correlate with occurrence and phenotypic consequences of mutations. Interestingly, cell-fate switches involve a transient usage of low ProT regulatory elements. Altogether, our work provides unprecedented insights into the gene regulatory landscape encoded in the DNA biophysical features.

CRISPR-Cas12a Nucleases Bind Flexible DNA Duplexes without RNA/DNA Complementarity.

Cas12a (also known as “Cpf1”) is a class 2 type V-A CRISPR-associated nuclease that can cleave double-stranded DNA at specific sites. The Cas12a effector enzyme comprises a single protein and a CRISPR-encoded small RNA (crRNA) and has been used for genome editing and manipulation. Work reported here examined in vitro interactions between the Cas12a effector enzyme and DNA duplexes with varying states of base-pairing between the two strands. The data revealed that in the absence of complementarity between the crRNA guide and the DNA target-strand, Cas12a binds duplexes with unpaired segments. These off-target duplexes were bound at the Cas12a site responsible for RNA-guided double-stranded DNA binding but were not cleaved due to the lack of RNA/DNA hybrid formation. Such promiscuous binding was attributed to increased DNA flexibility induced by the unpaired segment present next to the protospacer-adjacent-motif. The results suggest that target discrimination of Cas12a can be influenced by flexibility of the DNA. As such, in addition to the linear sequence, flexibility and other physical properties of the DNA should be considered in Cas12a-based genome engineering applications.

Human RAD52 Captures and Holds DNA Strands, Increases DNA Flexibility, and Prevents Melting of Duplex DNA: Implications for DNA Recombination

SummaryHuman RAD52 promotes annealing of complementary single-stranded DNA (ssDNA). In-depth knowledge of RAD52-DNA interaction is required to understand how its activity is integrated in DNA repair processes. Here, we visualize individual fluorescent RAD52 complexes interacting with single DNA molecules. The interaction with ssDNA is rapid, static, and tight, where ssDNA appears to wrap around RAD52 complexes that promote intra-molecular bridging. With double-stranded DNA (dsDNA), interaction is slower, weaker, and often diffusive. Interestingly, force spectroscopy experiments show that RAD52 alters the mechanics dsDNA by enhancing DNA flexibility and increasing DNA contour length, suggesting intercalation. RAD52 binding changes the nature of the overstretching transition of dsDNA and prevents DNA melting, which is advantageous for strand clamping during or after annealing. DNA-bound RAD52 is efficient at capturing ssDNA in trans. Together, these effects may help key steps in DNA repair, such as second-end capture during homologous recombination or strand annealing during RAD51-independent recombination reactions.

Probing Enhanced Double-Strand Break Formation at Abasic Sites within Clustered Lesions in Nucleosome Core Particles.

DNA is rapidly cleaved under mild alkaline conditions at apyrimidinic/apurinic sites, but the half-life is several weeks in phosphate buffer (pH 7.5). However, abasic sites are ∼100-fold more reactive within nucleosome core particles (NCPs). Histone proteins catalyze the strand scission, and at superhelical location 1.5, the histone H4 tail is largely responsible for the accelerated cleavage. The rate constant for strand scission at an abasic site is enhanced further in a nucleosome core particle when it is part of a bistranded lesion containing a proximal strand break. Cleavage of this form results in a highly deleterious double-strand break. This acceleration is dependent upon the position of the abasic lesion in the NCP and its structure. The enhancement in cleavage rate at an apurinic/apyrimidinic site rapidly drops off as the distance between the strand break and abasic site increases and is negligible once the two forms of damage are separated by 7 bp. However, the enhancement of the rate of double-strand break formation increases when the size of the gap is increased from one to two nucleotides. In contrast, the cleavage rate enhancement at 2-deoxyribonolactone within bistranded lesions is more modest, and it is similar in free DNA and nucleosome core particles. We postulate that the enhanced rate of double-strand break formation at bistranded lesions containing apurinic/apyrimidinic sites within nucleosome core particles is a general phenomenon and is due to increased DNA flexibility.

Effects of cytosine modifications on DNA flexibility and nucleosome mechanical stability.

Cytosine can undergo modifications, forming 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). Despite their importance as epigenetic markers and as central players in cellular processes, it is not well understood how these modifications influence physical properties of DNA and chromatin. Here we report a comprehensive survey of the effect of cytosine modifications on DNA flexibility. We find that even a single copy of 5-fC increases DNA flexibility markedly. 5-mC reduces and 5-hmC enhances flexibility, and 5-caC does not have a measurable effect. Molecular dynamics simulations show that these modifications promote or dampen structural fluctuations, likely through competing effects of base polarity and steric hindrance, without changing the average structure. The increase in DNA flexibility increases the mechanical stability of the nucleosome and vice versa, suggesting a gene regulation mechanism where cytosine modifications change the accessibility of nucleosomal DNA through their effects on DNA flexibility.

Hole Transfer Kinetics of DNA

Not long after the discovery of the double-helical structure of DNA in 1952, researchers proposed that charge transfer along a one-dimensional π-array of nucleobases might be possible. At the end of the 1990s researchers discovered that a positive charge (a hole) generated in DNA migrates more than 200 Å along the structure, a discovery that ignited interest in the charge-transfer process in DNA. As a result, DNA became an interesting potential bottom-up material for constructing nanoelectronic sensors and devices because DNA can form various complex two-dimensional and three-dimensional structures, such as smiley faces and cubes. From the fundamental aspects of the hole transfer process, DNA is one of the most well-studied organic molecules with many reports on the synthesis of artificial nucleobase analogues. Thus, DNA offers a unique system to study how factors such as the HOMO energy and molecular flexibility affect hole transfer kinetics. Understanding the hole transfer mechanism requires a discussion of the hole transfer rate constants (kHT). This Account reviews the kHT values determined by our group and by Lewis and Wasielewski's group, obtained by a combination of the synthesis of modified DNA and time-resolved spectroscopy. DNA consists of G/C and A/T base pairs; the HOMO localizes on the purine bases G and A, and G has a lower oxidation potential and a higher energy HOMO. Typically, long-range hole transfer proceeded via sequential hole transfer between G/C's. The kinetics of this process in DNA sequences, including those with mismatches, is reproducible via kinetic modeling using the determined kHT for each hole transfer step between G/C's. We also determined the distance dependence parameter (β), which describes the steepness of the exponential decrease of kHT. Because of this value, >0.6 Å(-1) for hole transfer in DNA, DNA itself does not serve as a molecular wire. Interestingly, hole transfer proceeded exceptionally fast for some sequences in which G/C's are located close to each other, an observation that we cannot explain by a simple sequential hole transfer between G/C's but rather through hole delocalization over the nucleobases. To further investigate and refine the factors that affect kHT, we examined various artificial nucleobases. We clearly demonstrated that kHT depends strongly on the HOMO energy gap between the bases (ΔHOMO), and that kHT can be increased with decreasing ΔHOMO. We reduced ΔHOMO between the two type of base pairs by replacing adenines (A's) with deazaadenines ((z)A's) or diaminopurines (D's) and showed that the hole transfer rate through the G/C and A/T mix sequence increased by more than 3 orders of magnitude. We also investigated how DNA flexibility affects kHT. Locked nucleic acid (LNA) modification, which makes DNA more rigid, lowered kHT by more than 2 orders of magnitude. On the other hand, 5-Me-2'-deoxyzebularine (B) modification, which increases DNA flexibility, increased kHT by more than 1 order of magnitude. These new insights in hole transfer kinetics obtained from modified DNAs may aid in the design of new molecular-scale conducting materials.

Single-molecule kinetics reveal microscopic mechanism by which High-Mobility Group B proteins alter DNA flexibility

Eukaryotic High-Mobility Group B (HMGB) proteins alter DNA elasticity while facilitating transcription, replication and DNA repair. We developed a new single-molecule method to probe non-specific DNA interactions for two HMGB homologs: the human HMGB2 box A domain and yeast Nhp6Ap, along with chimeric mutants replacing neutral N-terminal residues of the HMGB2 protein with cationic sequences from Nhp6Ap. Surprisingly, HMGB proteins constrain DNA winding, and this torsional constraint is released over short timescales. These measurements reveal the microscopic dissociation rates of HMGB from DNA. Separate microscopic and macroscopic (or local and non-local) unbinding rates have been previously proposed, but never independently observed. Microscopic dissociation rates for the chimeric mutants (∼10 s−1) are higher than those observed for wild-type proteins (∼0.1–1.0 s−1), reflecting their reduced ability to bend DNA through short-range interactions, despite their increased DNA-binding affinity. Therefore, transient local HMGB–DNA contacts dominate the DNA-bending mechanism used by these important architectural proteins to increase DNA flexibility.

Hole Transfer in LNA and 5-Me-2′-deoxyzebularine-Modified DNA

We report the measurement of hole-transfer rate constants (k(ht)) in locked nucleic acid (LNA) and 5-Me-2'-deoxyzebularine (B)-modified DNA. LNA modification, which makes DNA more rigid, caused a decrease of more than 2 orders of magnitude in k(ht), whereas B modification, which increases DNA flexibility, increased k(ht) by more than 20-fold. The present results clearly showed that hole-transfer efficiency in DNA can be increased by increasing DNA flexibility.

Effects of an Unnatural Base Pair Replacement on the Structure and Dynamics of DNA and Neighboring Water and Ions

Incorporating small molecule probes into biomolecular systems to report on local structure and dynamics is a powerful strategy that underlies a wide variety of experimental techniques, including fluorescence, electron paramagnetic resonance (EPR), and Forster resonance energy transfer (FRET) measurements. When an unnatural probe is inserted into a protein or DNA, the degree to which the presence of the probe has perturbed the local structure and dynamics it was intended to study is always an important concern. Here, molecular dynamics (MD) simulations are used to systematically study the effect of replacing a DNA base pair with a fluorescent probe, coumarin 102 deoxyriboside, at six unique sites along an A-tract DNA dodecamer. While the overall structure of the DNA oligonucleotide remains intact, replacement of A*T base pairs leads to widespread structural and dynamic perturbations up to four base pairs away from the probe site, including widening of the minor groove and increased DNA flexibility. New DNA conformations, not observed in the native sequence, are sometimes found in the vicinity of the probe and its partner abasic site analog. Strong correlations are demonstrated between DNA surface topology and water mobility.

HMGB Binding to DNA: Single and Double Box Motifs

High mobility group (HMG) proteins are nuclear proteins believed to significantly affect DNA interactions by altering nucleic acid flexibility. Group B (HMGB) proteins contain HMG box domains known to bind to the DNA minor groove without sequence specificity, slightly intercalating base pairs and inducing a strong bend in the DNA helical axis. A dual-beam optical tweezers system is used to extend double-stranded DNA (dsDNA) in the absence as well as presence of a single box derivative of human HMGB2 [HMGB2(box A)] and a double box derivative of rat HMGB1 [HMGB1(box A+box B)]. The single box domain is observed to reduce the persistence length of the double helix, generating sharp DNA bends with an average bending angle of 99±9° and, at very high concentrations, stabilizing dsDNA against denaturation. The double box protein contains two consecutive HMG box domains joined by a flexible tether. This protein also reduces the DNA persistence length, induces an average bending angle of 77±7°, and stabilizes dsDNA at significantly lower concentrations. These results suggest that single and double box proteins increase DNA flexibility and stability, albeit both effects are achieved at much lower protein concentrations for the double box. In addition, at low concentrations, the single box protein can alter DNA flexibility without stabilizing dsDNA, whereas stabilization at higher concentrations is likely achieved through a cooperative binding mode.

DNA twist, flexibility and transcription of the osmoregulated proU promoter of Salmonella typhimurium.

Transcription from many bacterial promoters is sensitive to the level of DNA supercoiling. We have investigated the mechanism by which environmentally induced changes in DNA supercoiling might regulate transcription. For the proU promoter of Salmonella typhimurium, osmotically induced changes in DNA topology appear to play a primary regulatory role. Changes in DNA supercoiling (linking number; delta Lk) are partitioned into changes in the winding of the strands of the double helix about themselves (twist; delta Tw) and/or elastic deformations or flexibility of the DNA helix (writhe; delta Wr). Mutations of the proU promoter were isolated in vivo, or generated in vitro, which altered the spacing between the -10 and -35 motifs. Studies on these mutant promoters, both in vivo and in vitro, exclude models in which changes in DNA twist play a regulatory role. Instead, our data suggest that increased DNA flexibility, reflecting the osmotically induced increase in negative supercoiling of DNA, is required for promoter activation.

MYB.Ph3 transcription factor from Petunia hybrida induces similar DNA‐bending/distortions on its two types of binding site

MYB.Ph3 from Petunia is a member of the MYB transcription factor family that recognizes two types of binding site, resembling direct and inverted repeats of the motif GTTA, respectively, and flanked on their 5' side by an A-rich sequence. In this study, it is shown that MYB.Ph3 induces similar conformational changes in both of its DNA-binding sites. Circular permutation assays indicate that the center of the distortions induced by MYB.Ph3 maps near the center of either MYB.Ph3-binding site. The degree of the distortion induced by MYB.Ph3 appears to be greatly affected by regions of the protein other than the DNA-binding domain, and differs from the distortion caused by animal c-MYB. Phasing analysis reveals that part of the distortion induced by MYB.Ph3 is DNA bending, oriented towards the minor groove, as is the case of its animal counterpart, c-MYB. DNA-binding by both MYB.Ph3 and animal c-MYB is more efficient in the presence of Ba2+, a divalent cation known to promote/stabilize DNA bending, than in the presence of other cations which do not favor this distortion, such as Na+ and Mg2+. In addition, both MYB proteins show higher affinity to selectively nicked DNA, which has increased DNA flexibility, strongly suggesting that DNA binding by these MYB proteins and DNA structural properties are mutually influenced.

Structural modification of DNA by a DNA-binding motif SPKK: detection of changes in base-pair hydrogen bonding and base stacking by UV resonance Raman spectroscopy.

Interactions of a DNA-binding motif SPKK with polynucleotides have been investigated by uv resonance Raman spectroscopy. Analysis of the Raman spectra has shown that the tetrapeptide SPKK weakens the adenine-thymine base-pair hydrogen bonding in poly(dA-dT).poly(dA-dT) and reduces the adenine-adenine base stacking interactions in poly(dA).poly(dT), both effects being indicative of destabilization of the DNA double helical structure. On the other hand, poly(dG-dC).poly(dG-dC) and poly(dG).poly(dC) do not show any structural change in the presence of SPKK. The present observations confirm that the SPKK motif, which is frequently found in histone H1 proteins, specifically binds to A/T-rich regions of DNA and loosens the DNA double-helical structure. One of the roles of the SPKK motifs in histones may be to increase DNA flexibility so that DNA can wrap around core histones and be assembled into chromosomes more easily.

DNA bending in the ternary nucleoprotein complex at the c-fos promoter.

Transcriptional induction of the c-fos proto-oncogene in response to serum growth factors is mediated in part by a ternary complex that forms on the serum response element (SRE) within its promoter. This complex consists of Elk-1, serum response factor (SRF) and the SRE. Elk-1 is phosphorylated by MAP kinase, which correlates with the induction of c-fos transcription. In this study we have investigated the protein-induced DNA bending which occurs during the formation and post-translational modification of the ternary complex that forms at the c-fos SRE. Circular permutation analysis demonstrates that the minimal DNA-binding domain of SRF, which contains the MADS box, is sufficient to induce flexibility into the centre of its binding site within the SRE. Phasing analysis indicates that at least part of this flexibility results in the production of a directional bend towards the minor groove. The isolated ETS domains from Elk-1 and SAP-1 induce neither DNA bending nor increased DNA flexibility. Formation of ternary complexes by binding of Elk-1 to the binary SRF:SRE complex results in a change in the flexibility of the SRE. Phosphorylation of Elk-1 by MAP kinase (p42/ERK2) induces further minor changes in this DNA flexibility. However, phasing analysis reveals that the recruitment of Elk-1 to form the ternary complex affects the SRF-induced directional DNA bend in the SRE. The potential roles of DNA bending at the c-fos SRE are discussed.

The maize chromosomal HMGa protein recognizes structural features of DNA and increases DNA flexibility.

The abundant maize high-mobility group protein HMGa belongs to the chromosomal, non-histone proteins and consists of a basic region containing the HMG-box DNA-binding domain and a highly acidic carboxy-terminal tail. The full-length HMGa protein and a truncated version lacking the acidic tail were synthesized in Escherichia coli and tested for their ability to induce DNA-bending in a ligase mediated circularization assay with short DNA fragments. It is shown that the recombinant HMGa protein as well as its truncated form efficiently cause circularization of the tested DNA fragments without an obvious requirement for stable DNA-binding. They bind furthermore preferentially to A/T-rich linear DNA or bent DNA structures such as four-way junctions and DNA minicircles. The DNA-binding properties and the ability to increase DNA flexibility suggest a general role of the HMGa protein in assisting the formation of nucleoprotein complexes, possibly by facilitating interactions of proteins bound to adjacent DNA sites.

The enhancer-blocking suppressor of Hairy-wing zinc finger protein of Drosophila melanogaster alters DNA structure.

Insertion of the gypsy retrotransposon of Drosophila melanogaster into a gene control region can repress gene expression. The zinc finger protein (SUHW) encoded by the suppressor of Hairy-wing [su(Hw)] gene binds to gypsy and prevents gene enhancers from activating transcription. SUHW blocks an enhancer only when positioned between the enhancer and promoter. Although position dependent, SUHW enhancer blocking is distance independent. These properties indicate that SUHW does not interact with the transcription activator proteins that bind to enhancers. To explore if DNA distortions are involved in enhancer blocking, the ability of SUHW to alter DNA structure was examined in gel mobility assays. Indeed, SUHW induces an unusual change in the structure of the binding-site DNA. The change is not a directed DNA bend but correlates with loss of sequence-directed bends in the unbound DNA. The DNA distortion requires a SUHW protein domain not required for DNA binding, and mutant proteins that fail to alter DNA structure also fail to eliminate the sequence-directed bends. These results suggest that SUHW increases DNA flexibility. The DNA distortion is not sufficient to block enhancers, and therefore it is suggested that increased DNA flexibility may help SUHW interact and interfere with proteins that support long-distance enhancer-promoter interactions.

The enhancer-blocking suppressor of Hairy-wing zinc finger protein of Drosophila melanogaster alters DNA structure.

Insertion of the gypsy retrotransposon of Drosophila melanogaster into a gene control region can repress gene expression. The zinc finger protein (SUHW) encoded by the suppressor of Hairy-wing [su(Hw)] gene binds to gypsy and prevents gene enhancers from activating transcription. SUHW blocks an enhancer only when positioned between the enhancer and promoter. Although position dependent, SUHW enhancer blocking is distance independent. These properties indicate that SUHW does not interact with the transcription activator proteins that bind to enhancers. To explore if DNA distortions are involved in enhancer blocking, the ability of SUHW to alter DNA structure was examined in gel mobility assays. Indeed, SUHW induces an unusual change in the structure of the binding-site DNA. The change is not a directed DNA bend but correlates with loss of sequence-directed bends in the unbound DNA. The DNA distortion requires a SUHW protein domain not required for DNA binding, and mutant proteins that fail to alter DNA structure also fail to eliminate the sequence-directed bends. These results suggest that SUHW increases DNA flexibility. The DNA distortion is not sufficient to block enhancers, and therefore it is suggested that increased DNA flexibility may help SUHW interact and interfere with proteins that support long-distance enhancer-promoter interactions.