The role of duplex ends in the spontaneous interaction of homologous linear DNA fragments

The spontaneous interaction of homologous linear DNA fragments was studied with a model of purified PCR products by agarose gel electrophoresis. To interact, duplexes required not only homology of internal regions, but also complementary ends. Fragments differing in terminal sequences did not interact. The yield of Holliday junctions (HJ), the simplest product of DNA-DNA interaction, depended on dissociation of fragment ends. Compared with genomic fragments, those with low-melting AT ends interacted with each other more efficiently and those with high-melting GC ends, less efficiently. Incubation temperature affected the equilibrium HJ concentration in solution of homologous fragments. A conclusion was made that HJ formation is initiated by nucleation of dissociated duplex ends.     

Mechanism of spontaneous DNA-DNA interaction of homologous linear duplexes

Previously, we demonstrated the interaction of homologous linear duplexes with formation of four-way DNA structures on the model of five PCR products. We propose that homologous duplex interaction is initiated by the nucleation of several dissociated base pairs of the complementary ends of two fragments with Holliday junction formation, in which cross point migration occurs via spooling of DNA strands from one duplex to the other one, finally resulting in complete resolution into new or previously existing duplexes. To confirm that DNA-DNA interaction involves formation of four-way DNA structures with strand exchange at the cross point, we have demonstrated the strand exchange process between identical duplexes using homologous fragments, harboring either biotin label or (32)P-label. Incubation of the mixture resulted in the addition of (32)P-label to biotin-labeled fragments, and the intensity of (32)P-labeling of biotinylated fragments was dependent upon the incubation duration. DNA-DNA interaction is not based on surface-dependent denaturing, as Triton X-100 does not decrease the formation of complexes between DNA duplexes. The equilibrium concentration of Holliday junctions depends on the sequences of the fragment ends and the incubation temperature. The free energy of Holliday junction formation by the fragments with GC and AT ends differed by 0.6 kcal/mol. Electron microscopic analysis demonstrated that the majority of Holliday junctions harbor the cross point within a 300 base pair region of the fragment ends. This insight into the mechanism of homologous duplex interaction extends our understanding of different DNA rearrangements. Understanding of DNA-DNA interaction is of practical use for better interpretation and optimization of PCR-based analyses.     

Position-specific interaction between cells of the imaginal wing and haltere discs of Drosophila melanogaster.

When fragments of the imaginal wing disc from opposite ends of the disc are mixed prior to culture, intercalary regeneration occurs so that structures are produced which neither of the fragments would have produced if they had been cultured alone. I report here that fragments of the imaginal wing and haltere disc interact in a position-specific way. Mixing of homologous fragments does not result in regeneration, while mixing of fragments from opposite ends of the discs does. Thus the interaction of wing and haltere disc fragments shows the same positional specificity as the mixing of two wing fragments.     

The RuvA homologues from Mycoplasma genitalium and Mycoplasma pneumoniae exhibit unique functional characteristics

The DNA recombination and repair machineries of Mycoplasma genitalium and Mycoplasma pneumoniae differ considerably from those of gram-positive and gram-negative bacteria. Most notably, M. pneumoniae is unable to express a functional RecU Holliday junction (HJ) resolvase. In addition, the RuvB homologues from both M. pneumoniae and M. genitalium only exhibit DNA helicase activity but not HJ branch migration activity in vitro. To identify a putative role of the RuvA homologues of these mycoplasmas in DNA recombination, both proteins (RuvA(Mpn) and RuvA(Mge), respectively) were studied for their ability to bind DNA and to interact with RuvB and RecU. In spite of a high level of sequence conservation between RuvA(Mpn) and RuvA(Mge) (68.8% identity), substantial differences were found between these proteins in their activities. First, RuvA(Mge) was found to preferentially bind to HJs, whereas RuvA(Mpn) displayed similar affinities for both HJs and single-stranded DNA. Second, while RuvA(Mpn) is able to form two distinct complexes with HJs, RuvA(Mge) only produced a single HJ complex. Third, RuvA(Mge) stimulated the DNA helicase and ATPase activities of RuvB(Mge), whereas RuvA(Mpn) did not augment RuvB activity. Finally, while both RuvA(Mge) and RecU(Mge) efficiently bind to HJs, they did not compete with each other for HJ binding, but formed stable complexes with HJs over a wide protein concentration range. This interaction, however, resulted in inhibition of the HJ resolution activity of RecU(Mge).     

Distinct Roles of Ape1 Protein,an Enzyme Involved in DNA Repair,in High or Low Linear Energy Transfer Ionizing Radiation-induced Cell Killing

High linear energy transfer (LET) radiation from space heavy charged particles or a heavier ion radiotherapy machine kills more cells than low LET radiation, mainly because high LET radiation-induced DNA damage is more difficult to repair. Relative biological effectiveness (RBE) is the ratio of the effects generated by high LET radiation to low LET radiation. Previously, our group and others demonstrated that the cell-killing RBE is involved in the interference of high LET radiation with non-homologous end joining but not homologous recombination repair. This effect is attributable, in part, to the small DNA fragments (≤40 bp) directly produced by high LET radiation, the size of which prevents Ku protein from efficiently binding to the two ends of one fragment at the same time, thereby reducing non-homologous end joining efficiency. Here we demonstrate that Ape1, an enzyme required for processing apurinic/apyrimidinic (known as abasic) sites, is also involved in the generation of small DNA fragments during the repair of high LET radiation-induced base damage, which contributes to the higher RBE of high LET radiation-induced cell killing. This discovery opens a new direction to develop approaches for either protecting astronauts from exposure to space radiation or benefiting cancer patients by sensitizing tumor cells to high LET radiotherapy.     

Activity and in vivo dynamics of Bacillus subtilis DisA are affected by RadA/Sms and by Holliday junction-processing proteins

Bacillus subtilis c-di-AMP synthase DisA and RecA-related RadA/Sms are involved in the repair of DNA damage in exponentially growing cells. We provide genetic evidence that DisA or RadA/Sms is epistatic to the branch migration translocase (BMT) RecG and the Holliday junction (HJ) resolvase RecU in response to DNA damage. We provide genetic evidence damage. Functional DisA-YFP formed dynamic foci in exponentially growing cells, which moved through the nucleoids at a speed compatible with a DNA-scanning mode. DisA formed more static structures in the absence of RecU or RecG than in wild type cells, while dynamic foci were still observed in cells lacking the BMT RuvAB. Purified DisA synthesizes c-di-AMP, but interaction with RadA/Sms or with HJ DNA decreases DisA-mediated c-di-AMP synthesis. RadA/Sms-YFP also formed dynamic foci in growing cells, but the foci moved throughout the cells rather than just on the nucleoids, and co-localized rarely with DisA-YFP foci, suggesting that RadA/Sms and DisA interact only transiently in unperturbed conditions. Our data suggest a model in which DisA moving along dsDNA indicates absence of DNA damage/replication stress via normal c-di-AMP levels, while interaction with HJ DNA/halted forks leads to reduced c-di-AMP levels and an ensuing block in cell proliferation. RadA/Sms may be involved in modulating DisA activities.     

Cloning of the mitochondrial genome of Anopheles quadrimaculatus

The entire 15 kilobase (kb) Anopheles quadrimaculatus mitochondrial DNA (mtDNA) was cloned as three EcoRI fragments in a bacteriophage vector and then subcloned into plasmid vectors. The cloned DNA was physically mapped with restriction endonucleases, and the maps were compared to the restriction patterns of native A. quadrimaculatus mtDNA. Several genes were mapped by sequencing the ends of A. quadrimaculatus mtDNA subclones and by hybridization with the previously characterized Aedes albopictus mtDNA clones. These portions of the genetic map were identical in gene order to those of Drosophila yakuba. The predicted amino acid sequence of the protein coding regions that were sequenced were between 72% and 98% homologous to D. yakuba. The cloned mtDNA will be useful as a probe for population genetic analysis of mosquitoes.     

Functional role of BLAP75 in BLM-topoisomerase IIIalpha-dependent holliday junction processing

The BLAP75 protein combines with the BLM helicase and topoisomerase (Topo) IIIalpha to form an evolutionarily conserved complex, termed the BTB complex, that functions to regulate homologous recombination. BLAP75 binds DNA, associates with both BLM and Topo IIIalpha, and enhances the ability of the BLM-Topo IIIalpha pair to branch migrate the Holliday junction (HJ) or dissolve the double Holliday junction (dHJ) structure to yield non-crossover recombinants. Here we seek to understand the relevance of the biochemical attributes of BLAP75 in HJ processing. With the use of a series of BLAP75 protein fragments, we show that the evolutionarily conserved N-terminal third of BLAP75 mediates complex formation with BLM and Topo IIIalpha and that the DNA binding activity resides in the C-terminal third of this novel protein. Interestingly, the N-terminal third of BLAP75 is just as adept as the full-length protein in the promotion of dHJ dissolution and HJ unwinding by BLM-Topo IIIalpha. Thus, the BLAP75 DNA binding activity is dispensable for the ability of the BTB complex to process the HJ in vitro. Lastly, we show that a BLAP75 point mutant (K166A), defective in Topo IIIalpha interaction, is unable to promote dHJ dissolution and HJ unwinding by BLM-Topo IIIalpha. This result provides proof that the functional integrity of the BTB complex is contingent upon the interaction of BLAP75 with Topo IIIalpha.     

DNA2 Cooperates with the WRN and BLM RecQ Helicases to Mediate Long-range DNA End Resection in Human Cells

The 5′-3′ resection of DNA ends is a prerequisite for the repair of DNA double strand breaks by homologous recombination, microhomology-mediated end joining, and single strand annealing. Recent studies in yeast have shown that, following initial DNA end processing by the Mre11-Rad50-Xrs2 complex and Sae2, the extension of resection tracts is mediated either by exonuclease 1 or by combined activities of the RecQ family DNA helicase Sgs1 and the helicase/endonuclease Dna2. Although human DNA2 has been shown to cooperate with the BLM helicase to catalyze the resection of DNA ends, it remains a matter of debate whether another human RecQ helicase, WRN, can substitute for BLM in DNA2-catalyzed resection. Here we present evidence that WRN and BLM act epistatically with DNA2 to promote the long-range resection of double strand break ends in human cells. Our biochemical experiments show that WRN and DNA2 interact physically and coordinate their enzymatic activities to mediate 5′-3′ DNA end resection in a reaction dependent on RPA. In addition, we present in vitro and in vivo data suggesting that BLM promotes DNA end resection as part of the BLM-TOPOIIIα-RMI1-RMI2 complex. Our study provides new mechanistic insights into the process of DNA end resection in mammalian cells.     

Assembly of nucleosome-like structures mediated by cauliflower DNA topoisomerase

Nucleosome-like structures have been efficiently assembled in vitro by interaction of cauliflower histones, pBR322 DNA and cauliflower DNA topoisomerase, as assayed by supercoiling of relaxed circular DNA and by digestion with micrococcal nuclease. The optimum ionic strength for supercoiling was 150 mM KCl and the optimum weight ratio of histone to DNA was approximately 1.0. Four histones, H2A, H2B, H3 and H4, were necessary for the optimum assembling conditions, and the nucleosomes assembled protected DNA fragments of approximately 150 bp in length. It was found that cauliflower DNA topoisomerase acts not only as a DNA-relaxing enzyme but also as a chaperon factor for nucleosome assembly.     

Restriction enzyme site-directed amplification PCR: a tool to identify regions flanking a marker DNA

An innovative combination of various recently described molecular methods was set up to efficiently identify regions flanking a marker DNA in insertional mutants of Chlamydomonas. The technique is named restriction enzyme site-directed amplification PCR (RESDA-PCR) and is based on the random distribution of frequent restriction sites in a genome and on a special design of primers. The primer design is based on the presence of a restriction site included in a low degenerated sequence at the 3' end and of a specific adapter sequence at the 5' end, with the two ends being linked by a polyinosine bridge. Specific primers of the marker DNA combined with the degenerated primers allow amplification of DNA fragments adjacent to the marker insertion by using two rounds of either short or long cycling procedures. Amplified fragments from 0.3 to 2 kb or more are routinely obtained at sufficient purity and quantity for direct sequencing. This method is fast, is reliable (87% success rate), and can be easily extrapolated to any organism and marker DNA by designing the appropriate primers. A procedure involving the PCR over enzyme digest fragments is also proposed for when, exceptionally, positive results are not obtained.     

Specific oligonucleotide invasion into the end of DNA duplex

Phenomenon of the interaction of a double-stranded DNA fragment with an oligonucleotide complementary to the end of the duplex strand was demonstrated to occur via formation of three-stranded DNA structure with an oligonucleotide invasion. It was shown that oligonucleotides complementary to the duplex ends inhibit Holliday junction formation in solutions of homologous linear DNA fragments. This effect depends on the oligonucleotide concentration, sequence and their complementarity to the duplex ends. Formation of three-stranded complexes was demonstrated using radiolabeled oligonucleotides by agarose gel-electrophoresis followed by autoradiography. Analysis of three-stranded DNA structures by chemical cleavage of non-canonical base pairs revealed that oligonucleotide invades into duplex ends via a sequential displacement mechanism and that the level of the invasion may vary considerably.     

Precise Sequential DNA Ligation on A Solid Substrate: Solid-Based Rapid Sequential Ligation of Multiple DNA Molecules

Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation.     

Partial purification and characterization of two types of homologous DNA pairing activity from rat testis nuclei

We describe the partial purification and characterization of two different types of homologous DNA pairing activity from rat testis nuclear extracts. The activities are separated from each other by single-stranded DNA-cellulose affinity chromatography. One activity requires single-stranded DNA ends and promotes the homologous pairing of single-stranded DNA fragments with double-stranded circular DNA and has an apparent molecular mass of 100 kDa as determined by gel filtration chromatography. This pairing activity does not require the addition of exogenous ATP and is strongly Mg²⁺-dependent. The second pairing activity promotes strand-transfer between single-stranded circular DNA and homologous double-stranded DNA fragments and has an apparent molecular mass of 30 kDa as determined by gel filtration chromatography. This pairing activity also does not require ATP but, in contrast to the former, is Mg²⁺-independent.     

Direct analysis of Holliday junction resolving enzyme in a DNA origami nanostructure

Holliday junction (HJ) resolution is a fundamental step for completion of homologous recombination. HJ resolving enzymes (resolvases) distort the junction structure upon binding and prior cleavage, raising the possibility that the reactivity of the enzyme can be affected by a particular geometry and topology at the junction. Here, we employed a DNA origami nano-scaffold in which each arm of a HJ was tethered through the base-pair hybridization, allowing us to make the junction core either flexible or inflexible by adjusting the length of the DNA arms. Both flexible and inflexible junctions bound to Bacillus subtilis RecU HJ resolvase, while only the flexible junction was efficiently resolved into two duplexes by this enzyme. This result indicates the importance of the structural malleability of the junction core for the reaction to proceed. Moreover, cleavage preferences of RecU-mediated reaction were addressed by analyzing morphology of the reaction products.     

Formation of a stable RuvA protein double tetramer is required for efficient branch migration in vitro and for replication fork reversal in vivo

In bacteria, RuvABC is required for the resolution of Holliday junctions (HJ) made during homologous recombination. The RuvAB complex catalyzes HJ branch migration and replication fork reversal (RFR). During RFR, a stalled fork is reversed to form a HJ adjacent to a DNA double strand end, a reaction that requires RuvAB in certain Escherichia coli replication mutants. The exact structure of active RuvAB complexes remains elusive as it is still unknown whether one or two tetramers of RuvA support RuvB during branch migration and during RFR. We designed an E. coli RuvA mutant, RuvA2(KaP), specifically impaired for RuvA tetramer-tetramer interactions. As expected, the mutant protein is impaired for complex II (two tetramers) formation on HJs, although the binding efficiency of complex I (a single tetramer) is as wild type. We show that although RuvA complex II formation is required for efficient HJ branch migration in vitro, RuvA2(KaP) is fully active for homologous recombination in vivo. RuvA2(KaP) is also deficient at forming complex II on synthetic replication forks, and the binding affinity of RuvA2(KaP) for forks is decreased compared with wild type. Accordingly, RuvA2(KaP) is inefficient at processing forks in vitro and in vivo. These data indicate that RuvA2(KaP) is a separation-of-function mutant, capable of homologous recombination but impaired for RFR. RuvA2(KaP) is defective for stimulation of RuvB activity and stability of HJ·RuvA·RuvB tripartite complexes. This work demonstrates that the need for RuvA tetramer-tetramer interactions for full RuvAB activity in vitro causes specifically an RFR defect in vivo.     

Effect of Single-Strand Break on Branch Migration and Folding Dynamics of Holliday Junctions

The Holliday junction (HJ), or four-way junction, is a central intermediate state of DNA for homologous genetic recombination and other genetic processes such as replication and repair. Branch migration is the process by which the exchange of homologous DNA regions occurs, and it can be spontaneous or driven by proteins. Unfolding of the HJ is required for branch migration. Our previous single-molecule fluorescence studies led to a model according to which branch migration is a stepwise process consisting of consecutive migration and folding steps. Folding of the HJ in one of the folded conformations terminates the branch migration phase. At the same time, in the unfolded state HJ rapidly migrates over entire homology region of the HJ in one hop. This process can be affected by irregularities in the DNA double helical structure, so mismatches almost terminate a spontaneous branch migration. Single-stranded breaks or nicks are the most ubiquitous defects in the DNA helix; however, to date, their effect on the HJ branch migration has not been studied. In addition, although nicked HJs are specific substrates for a number of enzymes involved in DNA recombination and repair, the role of this substrate specificity remains unclear. Our main goal in this work was to study the effect of nicks on the efficiency of HJ branch migration and the dynamics of the HJ. To accomplish this goal, we applied two single-molecule methods: atomic force microscopy and fluorescence resonance energy transfer. The atomic force microscopy data show that the nick does not prevent branch migration, but it does decrease the probability that the HJ will pass the DNA lesion. The single-molecule fluorescence resonance energy transfer approaches were instrumental in detailing the effects of nicks. These studies reveal a dramatic change of the HJ dynamics. The nick changes the structure and conformational dynamics of the junctions, leading to conformations with geometries that are different from those for the intact HJ. On the basis of these data, we propose a model of branch migration in which the propensity of the junction to unfold decreases the lifetimes of folded states, thereby increasing the frequency of junction fluctuations between the folded states.     

Solution Small Angle X-ray Scattering (SAXS) Studies of RecQ from Deinococcus radiodurans and Its Complexes with Junction DNA Substrates

RecQ helicases, essential enzymes for maintaining genome integrity, possess the capability to participate in a wide variety of DNA metabolisms. They can initiate the homologous recombination repair pathway by unwinding damaged dsDNA and suppress hyper-recombination by promoting Holliday junction (HJ) migration. To learn how DrRecQ participates in the homologous recombination repair pathway, solution structures of Deinococcus radiodurans RecQ (DrRecQ) and its complexes with DNA substrates were investigated by small angle x-ray scattering. We found that the catalytic core and the most N-terminal HRDC (helicase and RNase D C-terminal) domain (HRDC1) undergo a conformational change to a compact state upon binding to a junction DNA. Furthermore, models of DrRecQ in complexes with two kinds of junction DNA (fork junction and HJ) were built based on the small angle x-ray scattering data, and together with the EMSA results, possible binding sites were proposed. It is demonstrated that two DrRecQ molecules bind to the opposite arms of HJ. This architecture is similar to the RuvAB complex and is hypothesized to be highly conserved in the other HJ migration proteins. This work provides us new clues to understand the roles DrRecQ plays in the RecFOR pathway.     

Rad52 and Ku bind to different DNA structures produced early in double-strand break repair

DNA double-strand breaks are repaired by one of two main pathways, non-homologous end joining or homologous recombination. A competition for binding to DNA ends by Ku and Rad52, proteins required for non-homologous end joining and homologous recombination, respectively, has been proposed to determine the choice of repair pathway. In order to test this idea directly, we compared Ku and human Rad52 binding to different DNA substrates. How ever, we found no evidence that these proteins would compete for binding to the same broken DNA ends. Ku bound preferentially to DNA with free ends. Under the same conditions, Rad52 did not bind preferentially to DNA ends. Using a series of defined substrates we showed that it is single-stranded DNA and not DNA ends that were preferentially bound by Rad52. In addition, Rad52 aggregated DNA, bringing different single-stranded DNAs in close proximity. This activity was independent of the presence of DNA ends and of the ability of the single-stranded sequences to form extensive base pairs. Based on these DNA binding characteristics it is unlikely that Rad52 and Ku compete as ‘gatekeepers’ of different DNA double-strand break repair pathways. Rather, they interact with different DNA substrates produced early in DNA double-strand break repair.     

Quaternary structure and cleavage specificity of a poxvirus holliday junction resolvase

Recently, poxviruses were found to encode a protein with signature motifs present in the RuvC family of Holliday junction (HJ) resolvases, which have a key role in homologous recombination in bacteria. The vaccinia virus homolog A22 specifically cleaved synthetic HJ DNA in vitro and was required for the in vivo resolution of viral DNA concatemers into unit-length genomes with hairpin telomeres. It was of interest to further characterize a poxvirus resolvase in view of the low sequence similarity with RuvC, the absence of virus-encoded RuvA and RuvB to interact with, and the different functions of the viral and bacterial resolvases. Because purified A22 aggregated severely, studies were carried out with maltose-binding protein fused to A22 as well as to RuvC. Using gel filtration, chemical cross-linking, analytical ultracentrifugation, and light scattering, we demonstrated that A22 and RuvC are homodimers in solution. Furthermore, the dimeric form of the resolvase associated with HJ DNA, presumably facilitating the symmetrical cleavage of such structures. Like RuvC, A22 symmetrically cleaved fixed HJ junctions as well as junctions allowing strand mobility. Unlike RuvC and other members of the family, however, the poxvirus enzyme exhibited little cleavage sequence specificity. Structural and enzymatic similarities of poxvirus, bacterial, and fungal mitochondrial HJ resolvases are consistent with their predicted evolutionary relationship based on sequence analysis. The absence of a homologous resolvase in mammalian cells makes these microbial enzymes excellent potential therapeutic targets.