Annealing Of Complementary DNA Strands Research Articles

Biochemical characterization of the RNA-binding and RNA-DNA strand exchange activities of the human RAD52 protein.

RAD52 is a single-stranded DNA (ssDNA) binding protein that functions in the repair of DNA double-strand breaks (DSBs) by promoting the annealing of complementary DNA strands. RAD52 may also play an important role in an RNA transcript-dependent type of DSB repair, in which it reportedly binds to RNA and mediates the RNA-DNA strand exchange reaction. However, the mechanistic details of these functions are still unclear. In the present study, we utilized the domain fragments of RAD52 to biochemically characterize the single-stranded RNA (ssRNA) binding and RNA-DNA strand exchange activities of RAD52. We found that the N-terminal half of RAD52 is primarily responsible for both activities. By contrast, significant differences were observed for the roles of the C-terminal half in RNA-DNA and DNA-DNA strand exchange reactions. The C-terminal fragment stimulated the inverse RNA-DNA strand exchange activity displayed by the N-terminal fragment in trans, whereas the trans stimulatory effect by the C-terminal fragment was not observed in the inverse DNA-DNA or forward RNA-DNA strand exchange reactions. These results suggest the specific function of the C-terminal half of RAD52 in RNA-templated DSB repair.

Novel type of coagulation equation and its application to DNA repeat expansion process

DNA molecules containing repetitive motifs are prone to expand in their lengths. Once there appear a head to tail tandem of two identical DNA sequences in the system, they can propagate indefinitely by the mechanism involving cycles of staggered annealing of complementary DNA strands of variable lengths and polymerase mediated filling-in of the generated overhangs. Microgene Polymerization Reaction (MPR) is an experimental model for expansion of short repetitive DNA to longer lengths. The testable kinetic model of (MPR) was formulated and solved numerically by Itsko et al. in Kinetics of Repeat Propagation in the Microgene Polymerization Reaction (2009). Here, the simple cases of MPR were solved analytically using modified Smoluchowski coagulation equation. It was found that the repeats propagate according to Gumbel probability density function when the distribution of lengths of obtained polymers follows inverted Gumbel probability density function.

Yeast Pif1 accelerates annealing of complementary DNA strands.

Pif1 is a helicase involved in the maintenance of nuclear and mitochondrial genomes in eukaryotes. Here we report a new activity of Saccharomyces cerevisiae Pif1, annealing of complementary DNA strands. We identified preferred substrates for annealing as those that generate a duplex product with a single-stranded overhang relative to a blunt end duplex. Importantly, we show that Pif1 can anneal DNA in the presence of ATP and Mg2+. Pif1-mediated annealing also occurs in the presence of single-stranded DNA binding proteins. Additionally, we show that partial duplex substrates with 3′-single-stranded overhangs such as those generated during double-strand break repair can be annealed by Pif1.

Structural and Functional Characterization of the Redβ Recombinase from Bacteriophage λ

The Red system of bacteriophage λ is responsible for the genetic rearrangements that contribute to its rapid evolution and has been successfully harnessed as a research tool for genome manipulation. The key recombination component is Redβ, a ring-shaped protein that facilitates annealing of complementary DNA strands. Redβ shares functional similarities with the human Rad52 single-stranded DNA (ssDNA) annealing protein although their evolutionary relatedness is not well established. Alignment of Rad52 and Redβ sequences shows an overall low level of homology, with 15% identity in the N-terminal core domains as well as important similarities with the Rad52 homolog Sak from phage ul36. Key conserved residues were chosen for mutagenesis and their impact on oligomer formation, ssDNA binding and annealing was probed. Two conserved regions were identified as sites important for binding ssDNA; a surface basic cluster and an intersubunit hydrophobic patch, consistent with findings for Rad52. Surprisingly, mutation of Redβ residues in the basic cluster that in Rad52 are involved in ssDNA binding disrupted both oligomer formation and ssDNA binding. Mutations in the equivalent of the intersubunit hydrophobic patch in Rad52 did not affect Redβ oligomerization but did impair DNA binding and annealing. We also identified a single amino acid substitution which had little effect on oligomerization and DNA binding but which inhibited DNA annealing, indicating that these two functions of Redβ can be separated. Taken together, the results provide fresh insights into the structural basis for Redβ function and the important role of quaternary structure.

There and back again: new single-molecule insights in the motion of DNA repair proteins

Cellular DNA repair machines are constantly at work supporting the integrity of our genomes. Numerous proteins cooperate to form a complex and adaptive system dedicated to detection and timely processing of DNA damage. The molecular underpinnings of how these proteins locate and discriminate DNA lesions, match homologous sequences, mend the DNA and attend to a replication in distress are of a paramount biomedical importance, but in many cases remain unclear. Combined with more conventional tools, single-molecule biochemistry has been stepping in to address the age-old problems in the DNA repair field. This review will address new insights into diffusive properties of three DNA repair systems: I will discuss the emerging model of how MutS homologues locate and respond to mismatches in the dsDNA; the mechanism by which RAD52 promotes annealing of complementary DNA strands coated with ssDNA binding protein RPA; and how the nucleoprotein filament formed by RecA recombinase on ssDNA searches for homology within duplex DNA. These three distinct DNA repair factors exemplify the dynamic nature of cellular DNA repair machines revealed by single-molecule studies.

RAD52 variants predict platinum resistance and prognosis of cervical cancer.

RAD52 is an important but not well characterized homologous recombination repair gene that can bind to single-stranded DNA ends and mediate the DNA-DNA interaction necessary for the annealing of complementary DNA strands. To evaluate the role of RAD52 variants in the response of tumor cells to platinum agents, we investigated their associations with platinum resistance and prognosis in cervical cancer patients. We enrolled 154 patients with cervical squamous cell carcinoma, who had radical surgery between 2008 and 2009, and genotyped three potentially functional RAD52 variants by the SNaPshot assay. We tested in vitro platinum resistance and RAD52 expression by using the MTT and immunohistochemistry methods, respectively. In 144 cases who had genotyping data, we found that both the rs1051669 variant and RAD52 protein expression were significantly associated with carboplatin resistance (P = 0.024 and 0.028, respectively) and rs10774474 with nedaplatin resistance (P = 0.018). The rs1051669 variant was significantly associated with RAD52 protein expression (adjusted OR = 4.7, 95% CI = 1.4−16.1, P = 0.013). When these three RAD52 variants were combined, progression-free survival was lower in patients who carried at least one (≥1) variant allele compared to those without any of the variant alleles (P = 0.047). Therefore, both RAD52 variants and protein expression can predict platinum resistance, and RAD52 variants appeared to predict prognosis in cervical cancer patients. Large studies are warranted to validate these findings.

Enzymatic Activities and DNA Substrate Specificity of Mycobacterium tuberculosis DNA Helicase XPB

XPB, also known as ERCC3 and RAD25, is a 3′→5′ DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA) surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB), a 3′→5′ DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg2+/Mn2+. Consistent with the 3′→5′ polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3′ overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3′ DNA tail, it was not active on substrates containing a 3′ RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB.

Complex activities of the human Bloom's syndrome helicase are encoded in a core region comprising the RecA and Zn-binding domains

Bloom's syndrome DNA helicase (BLM), a member of the RecQ family, is a key player in homologous recombination (HR)-based error-free DNA repair processes. During HR, BLM exerts various biochemical activities including single-stranded (ss) DNA translocation, separation and annealing of complementary DNA strands, disruption of complex DNA structures (e.g. displacement loops) and contributes to quality control of HR via clearance of Rad51 nucleoprotein filaments. We performed a quantitative mechanistic analysis of truncated BLM constructs that are shorter than the previously identified minimal functional module. Surprisingly, we found that a BLM construct comprising only the two conserved RecA domains and the Zn2+-binding domain (residues 642–1077) can efficiently perform all mentioned HR-related activities. The results demonstrate that the Zn2+-binding domain is necessary for functional interaction with DNA. We show that the extensions of this core, including the winged-helix domain and the strand separation hairpin identified therein in other RecQ-family helicases, are not required for mechanochemical activity per se and may instead play modulatory roles and mediate protein–protein interactions.

RecO-mediated DNA homology search and annealing is facilitated by SsbA

Bacillus subtilis RecO plays a central role in recombinational repair and genetic recombination by (i) stimulating RecA filamentation onto SsbA-coated single-stranded (ss) DNA, (ii) modulating the extent of RecA-mediated DNA strand exchange and (iii) promoting annealing of complementary DNA strands. Here, we report that RecO-mediated strand annealing is facilitated by cognate SsbA, but not by a heterologous one. Analysis of non-productive intermediates reveals that RecO interacts with SsbA-coated ssDNA, resulting in transient ternary complexes. The self-interaction of ternary complexes via RecO led to the formation of large nucleoprotein complexes. In the presence of homology, SsbA, at the nucleoprotein, removes DNA secondary structures, inhibits spontaneous strand annealing and facilitates RecO loading onto SsbA–ssDNA complex. RecO relieves SsbA inhibition of strand annealing and facilitates transient and random interactions between homologous naked ssDNA molecules. Finally, both proteins lose affinity for duplex DNA. Our results provide a mechanistic framework for rationalizing protein release and dsDNA zippering as coordinated events that are crucial for RecA-independent plasmid transformation.

Kinetics of Repeat Propagation in the Microgene Polymerization Reaction

Repetitive DNA is a periodic copolymer with the intrinsic property of exponential propagation to longer repeats. Microgene polymerization reaction (MPR) is a model system in which a short nonrepetitive homo-duplex DNA evolves to multiple repetitive products during heat-cool cycles. The mechanism underlying this process involves staggered annealing of complementary DNA strands of variable lengths and polymerase-mediated filling-in of the generated overhangs. MPR is considered here as a process sharing common features with two polymerization types, chain-growth and step-growth, and significant distinctions from both types were highlighted. The involved reaction stages were formulated and a kinetic model was derived and tested experimentally. The model can quantitatively explain MPR propagation and be used as a good approximation for this phenomenon.

Functional and Structural Basis for a Bacteriophage Homolog of Human RAD52

In eukaryotes, homologous recombination proteins such as RAD51 and RAD52 play crucial roles in DNA repair and genome stability. Human RAD52 is a member of a large single-strand annealing protein (SSAP) family [1] and stimulates Rad51-dependent recombination [2, 3]. In prokaryotes and phages, it has been difficult to establish the presence of RAD52 homologs with conserved sequences. Putative SSAPs were recently found in several phages that infect strains of Lactococcus lactis[4]. One of these SSAPs was identified as Sak and was found in the virulent L. lactis phage ul36, which belongs to the Siphoviridae family [4, 5]. In this study, we show that Sak is homologous to the N terminus of human RAD52. Purified Sak binds single-stranded DNA (ssDNA) preferentially over double-stranded DNA (dsDNA) and promotes the renaturation of long complementary ssDNAs. Sak also binds RecA and stimulates homologous recombination reactions. Mutations shown to modulate RAD52 DNA binding [6] affect Sak similarly. Remarkably, electron-microscopic reconstruction of Sak reveals an undecameric (11) subunit ring, similar to the crystal structure of the N-terminal fragment of human RAD52 [7, 8]. For the first time, we propose a viral homolog of RAD52 at the amino acid, phylogenic, functional, and structural levels.

Specific DNA binding by the homeodomain Nkx2.5(C56S): Detection of impaired DNA or unfolded protein by isothermal titration calorimetry

Titrations of specific 18-bp duplex DNA with the cardiac-specific homeodomain Nkx2.5(C56S) have utilized an ultrasensitive isothermal titration calorimeter (ITC). As the free DNA nears depletion, we observe large apparent decreases in the binding enthalpy when the DNA is impaired or when the temperature is sufficiently high to produce some unfolding of the free protein. Either effect can be attributed to refolding of the biopolymer that occurs as a result of stabilization due to the large favorable change in free energy on the homeodomain binding to DNA (-49.4 kJ/mol at 298 K). In either case, thermodynamic parameters obtained in such ITC experiments are unreliable. By using a lower temperature (85 vs. 95 degrees C) during the annealing of complementary DNA strands, damage of the 18-bp duplex DNA (T(m) = 72 degrees C) is avoided, and titrations with the homeodomain are normal at temperatures from 10 to 40 degrees C when >95% of the protein is folded. Under the latter conditions, the heat capacity plot is linear with a DeltaC(p) value of -0.80 +/- 0.03 kJ K(-1) mol(-1), which is more negative than that calculated from the burial of solvent accessible surface areas (-0.64 +/- 0.05 kJ K(-1) mol(-1)), consistent with water structures being at the protein-DNA interfaces.

Structural and functional analysis of the baculovirus single-stranded DNA-binding protein LEF-3

The single-stranded DNA-binding protein LEF-3 of Autographa californica multinucleocapsid nucleopolyhedrovirus consists of 385 amino acid residues, forms oligomers, and promotes Mg2+-independent unwinding of DNA duplexes and annealing of complementary DNA strands. Partial proteolysis revealed that the DNA-binding domain of LEF-3 is located within a central region (residues 28 to 326) that is relatively resistant to proteolysis. In contrast, the N-terminus (27 residues) and C-terminal portion (59 residues) are not involved in interaction with DNA and are readily accessible to proteolytic digestion. Circular dichroism analyses showed that LEF-3 is a folded protein with an estimated α-helix content of more than 40%, but it is structurally unstable and undergoes unfolding in aqueous solutions at temperatures near 50 °C. Unfolding eliminated the LEF-3 domains that are resistant to proteolysis and randomized the digestion pattern by trypsin. The structural transition was irreversible and was accompanied by the generation of high molecular weight (MW) complexes. The thermal treatment inhibited DNA-binding and unwinding activity of LEF-3 but markedly stimulated its annealing activity. We propose that the shift in LEF-3 activities resulted from the generation of the high MW protein complexes, that specifically stimulate the annealing of complementary DNA strands by providing multiple DNA-binding sites and bringing into close proximity the interacting strands. The unfolded LEF-3 was active in a strand exchange reaction suggesting that it could be involved in the production of recombination intermediates.

The Redox State of the Baculovirus Single-stranded DNA-binding Protein LEF-3 Regulates Its DNA Binding, Unwinding, and Annealing Activities

The single-stranded (ss) DNA-binding protein LEF-3 of Autographa californica multinucleocapsid nucleopolyhedrovirus promoted Mg(2+)-independent unwinding of DNA duplexes and annealing of complementary DNA strands. The unwinding and annealing activities of LEF-3 appeared to act in a competitive manner and were determined by the ratio of protein to DNA. At subsaturating and saturating concentrations, LEF-3 promoted annealing, whereas it promoted unwinding at oversaturation of DNA substrates. The LEF-3 binding to ssDNA and unwinding activity were sensitive to redox agents and were inhibited by oxidation of thiol groups in LEF-3 with 1,1'-azobis(N,N-dimethylformamide) (diamide) or by modification with the thiol-conjugating agent N-ethylmaleimide. Both oxidation and alkylation increased the dissociation constant of the interaction with model oligonucleotides indicating a decrease in an intrinsic affinity of LEF-3 for ssDNA. These results proved that free thiol groups are essential both for LEF-3 interaction with ssDNA and for DNA unwinding. In contrast, oxidation or modification of thiol groups stimulated the annealing activity of LEF-3 partially due to suppression of its unwinding activity. Treatment of LEF-3 with the reducing agent dithiothreitol inhibited annealing, indicating association of this activity with the oxidized protein. Thus, the balance between annealing and unwinding activities of LEF-3 was determined by the redox state of protein with the oxidized state favoring annealing and the reduced state favoring unwinding. An LEF-3 mutant in which the conservative cysteine Cys(214) was replaced with serine showed both a decreased binding to DNA and a reduced unwinding activity, thus indicating that this residue might participate in the regulation of LEF-3 activities.

Nucleic acid chaperone activity of the ORF1 protein from the mouse LINE-1 retrotransposon.

Non-LTR retrotransposons such as L1 elements are major components of the mammalian genome, but their mechanism of replication is incompletely understood. Like retroviruses and LTR-containing retrotransposons, non-LTR retrotransposons replicate by reverse transcription of an RNA intermediate. The details of cDNA priming and integration, however, differ between these two classes. In retroviruses, the nucleocapsid (NC) protein has been shown to assist reverse transcription by acting as a "nucleic acid chaperone," promoting the formation of the most stable duplexes between nucleic acid molecules. A protein-coding region with an NC-like sequence is present in most non-LTR retrotransposons, but no such sequence is evident in mammalian L1 elements or other members of its class. Here we investigated the ORF1 protein from mouse L1 and found that it does in fact display nucleic acid chaperone activities in vitro. L1 ORF1p (i) promoted annealing of complementary DNA strands, (ii) facilitated strand exchange to form the most stable hybrids in competitive displacement assays, and (iii) facilitated melting of an imperfect duplex but stabilized perfect duplexes. These findings suggest a role for L1 ORF1p in mediating nucleic acid strand transfer steps during L1 reverse transcription.

Precise binding of single-stranded DNA termini by human RAD52 protein.

The human RAD52 protein, which exhibits a heptameric ring structure, has been shown to bind resected double strand breaks (DSBs), consistent with an early role in meiotic recombination and DSB repair. In this work, we show that RAD52 binds single-stranded and tailed duplex DNA molecules via precise interactions with the terminal base. When probed with hydroxyl radicals, ssDNA-RAD52 complexes exhibit a four-nucleotide repeat hypersensitivity pattern. This unique pattern is due to the interaction of RAD52 with either a 5' or a 3' terminus of the ssDNA, is sequence independent and is phased precisely from the terminal nucleotide. Hypersensitivity is observed over approximately 36 nucleotides, consistent with the length of DNA that is protected by RAD52 in nuclease protection assays. We propose that RAD52 binds DNA breaks via specific interactions with the terminal base, leading to the formation of a precisely organized ssDNA-RAD52 complex in which the DNA lies on an exposed surface of the protein. This protein-DNA arrangement may facilitate the DNA-DNA interactions necessary for RAD52-mediated annealing of complementary DNA strands.

Human 75-kDa DNA-pairing Protein Is Identical to the Pro-oncoprotein TLS/FUS and Is Able to Promote D-loop Formation

Homologous recombination plays a fundamental role in DNA double-strand break repair. Previously, we detected two mammalian nuclear proteins of 100 and 75 kDa (POMp100 and POMp75, respectively) that are able to promote homologous DNA pairing, a key step in homologous recombination. Here we describe the identification of human (h) POMp75 as the pro-oncoprotein TLS/FUS. hPOMp75/TLS binds both single- and double-stranded DNAs and mediates annealing of complementary DNA strands. More important, it promotes the uptake of a single-stranded oligonucleotide into a homologous superhelical DNA to form a D-loop. The formation of a D-loop is an essential step in DNA double-strand break repair through recombination. DNA annealing and D-loop formation catalyzed by hPOMp75/TLS require Mg(2+) and are ATP-independent. Interestingly, the oncogenic fusion form TLS-CHOP is not able to promote DNA pairing. These data suggest a possible role for hPOMp75/TLS in maintenance of genomic integrity.

Annealing of complementary DNA strands above the melting point of the duplex promoted by an archaeal protein

One enigma in the biology of hyperthermophilic microorganisms, living near or above 100°C, is how their genomes can be stable and, at the same time, plastic at temperatures above the melting point. The non-specific DNA-binding protein Sso7d of the hyperthermophilic archaeon Sulfolobus solfataricus is known to protect DNA from thermal denaturation. We report here that Sso7d promotes the renaturation of complementary DNA strands at temperatures above the melting point of the duplex. This novel annealing activity is strictly homology-dependent, and even one mismatch in a stretch of 17 complementary bases severely reduces its efficiency. Since pairing of homologous single strands is a key step in all fundamental processes involving nucleic acids, such as transcription, replication, recombination, and repair, Sso7d is a candidate component of the protein machinery devoted to the coupling of DNA stability to metabolic flexibility at high temperature.

RecA-mediated annealing of single-stranded DNA and its relation to the mechanism of homologous recombination

We demonstrate that RecA protein can mediate annealing of complementary DNA strands in vitro by at least two different mechanisms. The first annealing mechanism predominates under conditions where RecA protein causes coaggregation of single-stranded DNA (ssDNA) molecules and where RecA-free ssDNA stretches are present on both reaction partners. Under these conditions annealing can take place between locally concentrated protein-free complementary sequences. Other DNA aggregating agents like histone H1 or ethanol stimulate annealing by the same mechanism. The second mechanism of RecA-mediated annealing of complementary DNA strands is best manifested when preformed saturated RecA-ssDNA complexes interact with protein-free ssDNA. In this case, annealing can occur between the ssDNA strand resident in the complex and the ssDNA strand that interacts with the preformed RecA-ssDNA complex. Here, the action of RecA protein reflects its specific recombination promoting mechanism. This mechanism enables DNA molecules resident in the presynaptic RecA-DNA complexes to be exposed for hydrogen bond formation with DNA molecules contacting the presynaptic RecA-DNA filament.

RecA-mediated annealing of single-stranded DNA and its relation to the mechanism of homologous recombination*1

We demonstrate that RecA protein can mediate annealing of complementary DNA strands in vitro by at least two different mechanisms. The first annealing mechanism predominates under conditions where RecA protein causes coaggregation of single-stranded DNA (ssDNA) molecules and where RecA-free ssDNA stretches are present on both reaction partners. Under these conditions annealing can take place between locally concentrated protein-free complementary sequences. Other DNA aggregating agents like histone H1 or ethanol stimulate annealing by the same mechanism. The second mechanism of RecA-mediated annealing of complementary DNA strands is best manifested when preformed saturated RecA-ssDNA complexes interact with protein-free ssDNA. In this case, annealing can occur between the ssDNA strand resident in the complex and the ssDNA strand that interacts with the preformed RecA-ssDNA complex. Here, the action of RecA protein reflects its specific recombination promoting mechanism. This mechanism enables DNA molecules resident in the presynaptic RecA-DNA complexes to be exposed for hydrogen bond formation with DNA molecules contacting the presynaptic RecA-DNA filament.