Mechanism Of Strand Exchange Research Articles

TasA Fibre Interactions Are Necessary for Bacillus subtilis Biofilm Structure.

The extracellular matrix of biofilms provides crucial structural support to the community and protection from environmental perturbations. TasA, a key Bacillus subtilis biofilm matrix protein, forms both amyloid and non-amyloid fibrils. Non-amyloid TasA fibrils are formed via a strand-exchange mechanism, whereas the amyloid-like form involves non-specific self-assembly. We performed mutagenesis of the N-terminus to assess the role of non-amyloid fibrils in biofilm development. We find that the N-terminal tail is essential for the formation of structured biofilms, providing evidence that the strand-exchange fibrils are the active form in the biofilm matrix. Furthermore, we demonstrate that fibre formation alone is not sufficient to give structure to the biofilm. We build an interactome of TasA with other extracellular protein components, and identify important interaction sites. Our results provide insight into how protein-matrix interactions modulate biofilm development.

The strand exchange domain of tumor suppressor PALB2 is intrinsically disordered and promotes oligomerization-dependent DNA compaction.

The Partner and Localizer of BRCA2 (PALB2) is a scaffold protein that links BRCA1 with BRCA2 to initiate homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR efficiency in cells. The PALB2 DNA-binding domain (PALB2-DBD) supports strand exchange, a complex multistep reaction conducted by only a few proteins such as RecA-like recombinases and Rad52. Using bioinformatics analysis, small-angle X-ray scattering, circular dichroism, and electron paramagnetic spectroscopy, we determined that PALB2-DBD is an intrinsically disordered region (IDR) forming compact molten globule-like dimer. IDRs contribute to oligomerization synergistically with the coiled-coil interaction. Using confocal single-molecule FRET we demonstrated that PALB2-DBD compacts single-stranded DNA even in the absence of DNA secondary structures. The compaction is bimodal, oligomerization-dependent, and is driven by IDRs, suggesting a novel strand exchange mechanism. Intrinsically disordered proteins (IDPs) are prevalent in the human proteome. Novel DNA binding properties of PALB2-DBD and the complexity of strand exchange mechanism significantly expands the functional repertoire of IDPs. Multivalent interactions and bioinformatics analysis suggest that PALB2 function is likely to depend on formation of protein-nucleic acids condensates. Similar intrinsically disordered DBDs may use chaperone-like mechanism to aid formation and resolution of DNA and RNA multichain intermediates during DNA replication, repair and recombination.

Linear dichroism reveals the perpendicular orientation of DNA bases in the RecA and Rad51 recombinase filaments: A possible mechanism for the strand exchange reaction.

Linear dichroism spectroscopy is used to investigate the structure of RecA family recombinase filaments (RecA and Rad51 proteins) with DNA for clarifying the molecular mechanism of DNA strand exchange promoted by these proteins and its activation. The measurements show that the recombinases promote the perpendicular base orientation of single-stranded DNA only in the presence of activators, indicating the importance of base orientation in the reaction. We summarize the results and discuss the role of DNA base orientation.

Flanking strand separation activity of RecA nucleoprotein filaments in DNA strand exchange reactions.

The recombinase RecA/Rad51 ATPase family proteins catalyze paramount DNA strand exchange reactions that are critically involved in maintaining genome integrity. However, it remains unclear how DNA strand exchange proceeds when encountering RecA-free defects in recombinase nucleoprotein filaments. Herein, by designing a series of unique substrates (e.g. truncated or conjugated incoming single-stranded DNA, and extended donor double-stranded DNA) and developing a two-color alternating excitation-modified single-molecule real-time fluorescence imaging assay, we resolve the two key steps (donor strand separation and new base-pair formation) that are usually inseparable during the reaction, revealing a novel long-range flanking strand separation activity of synaptic RecA nucleoprotein filaments. We further evaluate the kinetics and free energetics of strand exchange reactions mediated by various substrates, and elucidate the mechanism of flanking strand separation. Based on these findings, we propose a potential fundamental molecular model involved in flanking strand separation, which provides new insights into strand exchange mechanism and homologous recombination.

The RecA-directed recombination pathway of natural transformation initiates at chromosomal replication forks in the pneumococcus

Homologous recombination (HR) is a crucial mechanism of DNA strand exchange that promotes genetic repair and diversity in all kingdoms of life. Bacterial HR is driven by the universal recombinase RecA, assisted in the early steps by dedicated mediators that promote its polymerization on single-stranded DNA (ssDNA). In bacteria, natural transformation is a prominent HR-driven mechanism of horizontal gene transfer specifically dependent on the conserved DprA recombination mediator. Transformation involves internalization of exogenous DNA as ssDNA, followed by its integration into the chromosome by RecA-directed HR. How DprA-mediated RecA filamentation on transforming ssDNA is spatiotemporally coordinated with other cellular processes remains unknown. Here, we tracked the localization of fluorescent fusions to DprA and RecA in Streptococcus pneumoniae and revealed that both accumulate in an interdependent manner with internalized ssDNA at replication forks. In addition, dynamic RecA filaments were observed emanating from replication forks, even with heterologous transforming DNA, which probably represent chromosomal homology search. In conclusion, this unveiled interaction between HR transformation and replication machineries highlights an unprecedented role for replisomes as landing pads for chromosomal access of tDNA, which would define a pivotal early HR step for its chromosomal integration.

Type V Pilus is Assembled by a Protease-mediated Strand-exchange Mechanism

Type V Pilus is Assembled by a Protease-mediated Strand-exchange Mechanism

Requirements for efficient ligand-gated co-transcriptional switching in designed variants of the B. subtilis pbuE adenine-responsive riboswitch in E. coli.

Riboswitches, generally located in the 5’-leader of bacterial mRNAs, direct expression via a small molecule-dependent structural switch that informs the transcriptional or translational machinery. While the structure and function of riboswitch effector-binding (aptamer) domains have been intensely studied, only recently have the requirements for efficient linkage between small molecule binding and the structural switch in the cellular and co-transcriptional context begun to be actively explored. To address this aspect of riboswitch function, we have performed a structure-guided mutagenic analysis of the B. subtilis pbuE adenine-responsive riboswitch, one of the simplest riboswitches that employs a strand displacement switching mechanism to regulate transcription. Using a cell-based fluorescent protein reporter assay to assess ligand-dependent regulatory activity in E. coli, these studies revealed previously unrecognized features of the riboswitch. Within the aptamer domain, local and long-range conformational dynamics influenced by sequences within helices have a significant effect upon efficient regulatory switching. Sequence features of the expression platform including the pre-aptamer leader sequence, a toehold helix and an RNA polymerase pause site all serve to promote strong ligand-dependent regulation. By optimizing these features, we were able to improve the performance of the B. subtilis pbuE riboswitch in E. coli from 5.6-fold induction of reporter gene expression by the wild type riboswitch to over 120-fold in the top performing designed variant. Together, these data point to sequence and structural features distributed throughout the riboswitch required to strike a balance between rates of ligand binding, transcription and secondary structural switching via a strand exchange mechanism and yield new insights into the design of artificial riboswitches.

Mechanism of strand exchange from RecA-DNA synaptic and D-loop structures.

The strand exchange reaction is central to homologous recombination. It is catalyzed by the RecA family of ATPases that form a helical filament with single-stranded DNA (ssDNA) and ATP. This filament binds to a donor double-stranded DNA (dsDNA) to form synaptic filaments that search for homology, and then catalyze the exchange of the complementary strand to form a new heteroduplex, or a D-loop if homology is limited1,2. Here we report the Cryo-EM analysis of synaptic mini filaments with both non-complementary and partially-complementary dsDNA, and structures of RecA–D-loop complexes containing a 10 or 12 base pair heteroduplex at 2.8 and 2.9 Å, respectively. The RecA C-terminal domain (CTD) binds to dsDNA and directs it to the L2 loop, which inserts into and opens the duplex. The opening propagates through RecA sequestering the homologous strand at a secondary DNA-binding site, freeing the complementary strand to sample pairing with the ssDNA. Duplex opening has a significant probability of stopping at each RecA step, with the as yet unopened dsDNA portion binding to another CTD. Homology suppresses this process through heteroduplex pairing cooperating with secondary site-ssDNA binding to extend dsDNA opening. This mechanism locally limits the length of ssDNA sampled for pairing if homology is not encountered, and it may provide for the formation of multiple synapses separated substantially on the donor dsDNA, increasing the probability of encountering homology.

Biogenesis of Type V pili.

Pili or fimbriae, which are filamentous structures present on the surface of bacteria, were purified from a periodontal pathogen, Porphyromonas gingivalis, in 1980s. The protein component of pili (stalk pilin), which is its major component, was named FimA; it has a molecular weight of approximately 41 kDa. Because the molecular weight of the pilin from P. gingivalis is twice that of pilins from other bacterial pili, the P. gingivalis Fim pili were suggested to be formed via a novel mechanism. In earlier studies, we reported that the FimA pilin is secreted on the cell surface as a lipoprotein precursor, and the subsequent N-terminal processing of the FimA precursor by arginine-specific proteases is necessary for Fim pili formation. The crystal structures of FimA and its related proteins were determined recently, which show that Fim pili are formed by a protease-mediated strand-exchange mechanism. The most recent study conducted by us, wherein we performed cryoelectron microscopy of the pilus structure, provided evidence in support of this mechanism. As the P. gingivalis Fim pili are formed through novel transport and assembly mechanisms, such pili are now designated as Type V pili. Surface lipoproteins, including the anchor pilin FimB of Fim pili that are present on the outer membrane, have been detected in certain Gram-negative bacteria. Here, we describe the assembly mechanisms of pili, including those of Type V and other pili, as well as the lipoprotein transport mechanisms.

Transport and Polymerization of Porphyromonas gingivalis Type V Pili.

Adhesive pili (or fimbriae) in bacteria are classified into five types, among which type V pili have been most recently described. Type V pili differ from other pili types with respect to transport mechanism, structure, and pilin synthesis. Genes of type V pili are restricted to the phylum Bacteroidetes. Protein subunits that compose type V pili are transported to the cell surface as lipoprotein precursors and then polymerized into a pilus through a strand-exchange mechanism, which is demonstrated by several experiments, including palmitic acid labeling and Cys-Cys cross-linking analysis. Here, we describe the use of these methods to analyze type V pili.

Loop-closure kinetics reveal a stable, right-handed DNA intermediate in Cre recombination.

In Cre site-specific recombination, the synaptic intermediate is a recombinase homotetramer containing a pair of loxP DNA target sites. The enzyme system's strand-exchange mechanism proceeds via a Holliday-junction (HJ) intermediate; however, the geometry of DNA segments in the synapse has remained highly controversial. In particular, all crystallographic structures are consistent with an achiral, planar Holliday-junction (HJ) structure, whereas topological assays based on Cre-mediated knotting of plasmid DNAs are consistent with a right-handed chiral junction. We use the kinetics of loop closure involving closely spaced (131–151 bp) loxP sites to investigate the in-aqueo ensemble of conformations for the longest-lived looped DNA intermediate. Fitting the experimental site-spacing dependence of the loop-closure probability, J, to a statistical-mechanical theory of DNA looping provides evidence for substantial out-of-plane HJ distortion, which unequivocally stands in contrast to the square-planar intermediate geometry from Cre-loxP crystal structures and those of other int-superfamily recombinases. J measurements for an HJ-isomerization-deficient Cre mutant suggest that the apparent geometry of the wild-type complex is consistent with temporal averaging of right-handed and achiral structures. Our approach connects the static pictures provided by crystal structures and the natural dynamics of macromolecules in solution, thus advancing a more comprehensive dynamic analysis of large nucleoprotein structures and their mechanisms.

CtDNA Detection Based on DNA Clutch Probes and Strand Exchange Mechanism.

Circulating tumor DNA (ctDNA), originating directly from the tumor or circulating tumor cells, may reflect the entire tumor genom and has gained considerable attention for its potential clinical diagnosis and prognosis throughout the treatment regimen. However, the reliable and robust ctDNA detection remains a key challenge. Here, this work designs a pair of DNA clutch separation probes and an ideal discrimination probes based on toehold-mediated strand displacement reaction (TSDR) to specifically recognize ctDNA. First, the ctDNAs were denatured to form ssDNAs, the pair of DNA clutch separation probes [one of which modified onto the magnetic nanoparticles (MNPs)] are used to recognize and hybridize with the complemental chains and prevent reassociation of denatured ssDNAs. The complemental chains are removed in magnetic field and left the wild and mutant ssDNA chains in the supernatant. Then, the TSDR specificity recognizes the target mutant sequence to ensure only the mutated strands to be detection. The proposed assay exhibited good sensitivity and selectivity without any signal amplification. The proposed assay displayed a linear range from 2 to100 nM with a limit of detection (LOD) of 0.85 nM, and it was useful for ctDNA biomedical analysis and clinic theranostic.

Bubble-bound state of triple-stranded DNA: Efimov physics in DNA with repulsion

The presence of a thermodynamic phase of a three-stranded DNA, namely, a mixed phase of bubbles of two bound strands and a single one, is established for large dimensions () by using exact real space renormalization group transformations and exact computations of specific heat for finite length chains. Similar exact computations for the fractal Sierpinski gasket of dimension d < 2 establish the stability of the phase in the presence of a repulsive three chain interaction. Although, for d < 2, cooperativity factors for bubbles or noncrossing conditions are needed for the melting transition, the mixed phase may exist even in absence of those. In contrast to the Efimov DNA, where three strands are bound though no two are bound, the mixed phase appears at temperatures less than the two chain melting temperature. Both the Efimov-DNA and the mixed phase are formed essentially due to the strand exchange mechanism.

A time for promiscuity in a eukaryotic recombinase

The exchange of DNA strands between broken and intact molecules lies at the heart of fundamental cellular processes ranging from repairing DNA damage by homologous recombination to generation of genetic diversity during sexual reproduction. New work by Lee and colleagues utilizes the DNA curtain method, an elegant single-molecule technique, to demonstrate common and idiosyncratic features in the DNA strand exchange mechanisms of three RecA-family recombinases, bacterial RecA, and eukaryotic Rad51 and Dmc1 proteins.

Iterative homology checking and non-uniform stepping during RecA-mediated strand exchange

Recombinase-mediated homologous recombination (HR) in which strands are exchanged between two similar or identical DNA molecules is essential for maintaining genome fidelity and generating genetic diversity. It is believed that HR comprises two distinct stages: an initial alignment with stringent homology checking followed by stepwise heteroduplex expansion. If and how homology checking takes place during heteroduplex expansion, however, remains unknown. In addition, the number of base pairs (bp) involved in each step is still under debate. By using single-molecule approaches to catch transient intermediates in RecA-mediated HR with different degrees of homology, we show that (i) the expansion proceeds with step sizes of multiples of 3 bp, (ii) the step sizes follow wide distributions that are similar to that of initial alignment lengths, and (iii) each distribution can be divided into a short-scale and a long-scale part irrespective of the degree of homology. Our results suggest an iterative mechanism of strand exchange in which ssDNA-RecA filament interrogates double-stranded DNA using a short tract (6–15 bp) for quick checking and a long tract (>18 bp) for stringent sequence comparison. The present work provides novel insights into the physical and structural bases of DNA recombination.

A Distinct Type of Pilus from the Human Microbiome

Pili are proteinaceous polymers of linked pilins that protrude from the cell surface of many bacteria and often mediate adherence and virulence. We investigated a set of 20 Bacteroidia pilins from the human microbiome whose structures and mechanism of assembly were unknown. Crystal structures and biochemical data revealed a diverse protein superfamily with a common Greek-key β sandwich fold with two transthyretin-like repeats that polymerize into a pilus through a strand-exchange mechanism. The assembly mechanism of the central, structural pilins involves proteinase-assisted removal of their N-terminal β strand, creating an extended hydrophobic groove that binds the C-terminal donor strands of the incoming pilin. Accessory pilins at the tip and base have unique structural features specific to their location, allowing initiation or termination of the assembly. The Bacteroidia pilus, therefore, has a biogenesis mechanism that is distinct from other known pili and likely represents a different type of bacterial pilus.

Controlled rotation mechanism of DNA strand exchange by the Hin serine recombinase.

DNA strand exchange by serine recombinases has been proposed to occur by a large-scale rotation of halves of the recombinase tetramer. Here we provide the first direct physical evidence for the subunit rotation mechanism for the Hin serine invertase. Single-DNA looping assays using an activated mutant (Hin-H107Y) reveal specific synapses between two hix sites. Two-DNA “braiding” experiments, where separate DNA molecules carrying a single hix are interwound, show that Hin-H107Y cleaves both hix sites and mediates multi-step rotational relaxation of the interwinding. The variable numbers of rotations in the DNA braid experiments are in accord with data from bulk experiments that follow DNA topological changes accompanying recombination by the hyperactive enzyme. The relatively slow Hin rotation rates, combined with pauses, indicate considerable rotary friction between synapsed subunit pairs. A rotational pausing mechanism intrinsic to serine recombinases is likely to be crucial for DNA ligation and for preventing deleterious DNA rearrangements.

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3' end of the initiating DNA strand have a small effect, whereas most mismatches near the 5' end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity.

Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE.

A crucial component of the human mitochondrial DNA replisome is the ring-shaped helicase TWINKLE—a phage T7-gene 4-like protein expressed in the nucleus and localized in the human mitochondria. Our previous studies showed that despite being a helicase, TWINKLE has unique DNA annealing activity. At the time, the implications of DNA annealing by TWINKLE were unclear. Herein, we report that TWINKLE uses DNA annealing function to actively catalyze strand-exchange reaction between the unwinding substrate and a homologous single-stranded DNA. Using various biochemical experiments, we demonstrate that the mechanism of strand-exchange involves active coupling of unwinding and annealing reactions by the TWINKLE. Unlike strand-annealing, the strand-exchange reaction requires nucleotide hydrolysis and greatly stimulated by short region of homology between the recombining DNA strands that promote joint molecule formation to initiate strand-exchange. Furthermore, we show that TWINKLE catalyzes branch migration by resolving homologous four-way junction DNA. These four DNA modifying activities of TWINKLE: strand-separation, strand-annealing, strand-exchange and branch migration suggest a dual role of TWINKLE in mitochondrial DNA maintenance. In addition to playing a major role in fork progression during leading strand DNA synthesis, we propose that TWINKLE is involved in recombinational repair of the human mitochondrial DNA.

Serine Resolvases.

Serine resolvases are an interesting group of site-specific recombinases that, in their native contexts, resolve large fused replicons into smaller separated ones. Some resolvases are encoded by replicative transposons and resolve the transposition product, in which the donor and recipient molecules are fused, into separate replicons. Other resolvases are encoded by plasmids and function to resolve plasmid dimers into monomers. Both types are therefore involved in the spread and maintenance of antibiotic-resistance genes. Resolvases and the closely related invertases were the first serine recombinases to be studied in detail, and much of our understanding of the unusual strand exchange mechanism of serine recombinases is owed to those early studies. Resolvases and invertases have also served as paradigms for understanding how DNA topology can be harnessed to regulate enzyme activity. Finally, their relatively modular structure, combined with a wealth of structural and biochemical data, has made them good choices for engineering chimeric recombinases with designer specificity. This chapter focuses on the current understanding of serine resolvases, with a focus on the contributions of structural studies.