Single-stranded DNA Ends Research Articles

Polynucleotide phosphorylase exonuclease and polymerase activities on single-stranded DNA ends are modulated by RecN, SsbA and RecA proteins

Bacillus subtilis pnpA gene product, polynucleotide phosphorylase (PNPase), is involved in double-strand break (DSB) repair via homologous recombination (HR) or non-homologous end-joining (NHEJ). RecN is among the first responders to localize at the DNA DSBs, with PNPase facilitating the formation of a discrete RecN focus per nucleoid. PNPase, which co-purifies with RecA and RecN, was able to degrade single-stranded (ss) DNA with a 3′ → 5′ polarity in the presence of Mn2+ and low inorganic phosphate (Pi) concentration, or to extend a 3′-OH end in the presence dNDP·Mn2+. Both PNPase activities were observed in evolutionarily distant bacteria (B. subtilis and Escherichia coli), suggesting conserved functions. The activity of PNPase was directed toward ssDNA degradation or polymerization by manipulating the Pi/dNDPs concentrations or the availability of RecA or RecN. In its dATP-bound form, RecN stimulates PNPase-mediated polymerization. ssDNA phosphorolysis catalyzed by PNPase is stimulated by RecA, but inhibited by SsbA. Our findings suggest that (i) the PNPase degradative and polymerizing activities might play a critical role in the transition from DSB sensing to end resection via HR and (ii) by blunting a 3′-tailed duplex DNA, in the absence of HR, B. subtilis PNPase might also contribute to repair via NHEJ.

Characterization of the Endonuclease and ATP-dependent Flap Endo/Exonuclease of Dna2

Two processes, DNA replication and DNA damage repair, are key to maintaining genomic fidelity. The Dna2 enzyme lies at the heart of both of these processes, acting in conjunction with flap endonuclease 1 and replication protein A in DNA lagging strand replication and with BLM/Sgs1 and MRN/X in double strand break repair. In vitro, Dna2 helicase and flap endo/exonuclease activities require an unblocked 5' single-stranded DNA end to unwind or cleave DNA. In this study we characterize a Dna2 nuclease activity that does not require, and in fact can create, 5' single-stranded DNA ends. Both endonuclease and flap endo/exonuclease are abolished by the Dna2-K677R mutation, implicating the same active site in catalysis. In addition, we define a novel ATP-dependent flap endo/exonuclease activity, which is observed only in the presence of Mn(2+). The endonuclease is blocked by ATP and is thus experimentally distinguishable from the flap endo/exonuclease function. Thus, Dna2 activities resemble those of RecB and AddAB nucleases even more closely than previously appreciated. This work has important implications for understanding the mechanism of action of Dna2 in multiprotein complexes, where dissection of enzymatic activities and cofactor requirements of individual components contributing to orderly and precise execution of multistep replication/repair processes depends on detailed characterization of each individual activity.

Double-Strand Breaks in Heterochromatin Move Outside of a Dynamic HP1a Domain to Complete Recombinational Repair

Double-strand breaks (DSBs) in heterochromatic repetitive DNAs pose significant threats to genome integrity, but information about how such lesions are processed and repaired is sparse. We observe dramatic expansion and dynamic protrusions of the heterochromatin domain in response to ionizing radiation (IR) in Drosophila cells. We also find that heterochromatic DSBs are repaired by homologous recombination (HR) but with striking differences from euchromatin. Proteins involved in early HR events (resection) are rapidly recruited to DSBs within heterochromatin. In contrast, Rad51, which mediates strand invasion, only associates with DSBs that relocalize outside of the domain. Heterochromatin expansion and relocalization of foci require checkpoint and resection proteins. Finally, the Smc5/6 complex is enriched in heterochromatin and is required to exclude Rad51 from the domain and prevent abnormal recombination. We propose that the spatial and temporal control of DSB repair in heterochromatin safeguards genome stability by preventing aberrant exchanges between repeats.

Purification, crystallization and preliminary X-ray diffraction analysis of DNA damage response A protein from Deinococcus radiodurans.

DNA damage response A protein (DdrA) from Deinococcus radiodurans has been suggested to be involved in DNA-repair processes through binding to 3'-ends of single-stranded DNA, thereby protecting the ends from nuclease digestion. In this study, a recombinant C-terminally truncated form of D. radiodurans DdrA (DdrA157) comprising the first 157 residues of DdrA was expressed in Escherichia coli, purified and crystallized. Single crystals of DdrA157 were obtained by the hanging-drop method at 293 K. The crystal belonged to the monoclinic space group P2(1), with unit-cell parameters a=46.31, b=180.26, c=114.17 Å, β=90.02°. The crystal was expected to contain 14 molecules in the asymmetric unit. Diffraction data were collected to 2.35 Å resolution on beamline BL-5 at Photon Factory and initial phase determinations were attempted by the molecular-replacement method using the human Rad52 structure.

Molecular recognition determinants for type IV secretion of diverse families of conjugative relaxases

In preparation for transfer conjugative type IV secretion systems (T4SS) produce a nucleoprotein adduct containing a relaxase enzyme covalently linked to the 5' end of single-stranded plasmid DNA. The bound relaxase is expected to present features necessary for selective recognition by the type IV coupling protein (T4CP), which controls substrate entry to the envelope spanning secretion machinery. We prove that the IncF plasmid R1 relaxase TraI is translocated to the recipient cells. Using a Cre recombinase assay (CRAfT) we mapped two internally positioned translocation signals (TS) on F-like TraI proteins that independently mediate efficient recognition and secretion. Tertiary structure predictions for the TS matched best helicase RecD2 from Deinococcus radiodurans. The TS is widely conserved in MOB(F) and MOB(Q) families of relaxases. Structure/function relationships within the TS were identified by mutation. A key residue in specific recognition by T4CP TraD was revealed by a fidelity switch phenotype for an F to plasmid R1 exchange L626H mutation. Finally, we show that physical linkage of the relaxase catalytic domain to a TraI TS is necessary for efficient conjugative transfer.

Comparison of the molecular influences of NO-induced lesions in DNA strands on the reactivity of polynucleotide kinases, DNA ligases and DNA polymerases

Nitric oxide (NO) causes DNA damage, generating xanthine (Xan, X) and oxanine (Oxa, O) from guanine (Gua, G) and hypoxanthine (Hyp, H) from adenine (Ade, A) by nitrosative oxidation. Although these NO-induced lesions have been thought to cause mutagenic problems in cellular systems, the influence of these lesions on enzymatic functions has not yet been compared systematically. In this study, we investigated the effect of NO-induced lesions on the activities of DNA-binding/recognizing enzymes such as T4 polynucleotide kinase (T4 PNK), DNA ligases (T4 DNA ligase, Taq DNA ligase) and DNA polymerases (E. coli DNA polymerase I, Klenow fragment, T4 DNA polymerase). The phosphorylation efficiencies of T4 PNK are dependent on the base type at the 5'-end of single-stranded DNA, where Oxa congruent with Hyp congruent with Gua > Xan congruent with Ade. The enzymatic reactions efficiencies of DNA ligases or DNA polymerases were observed to be dependent on the base-pairing type bound by the enzymes, where G:C > H:C > O:C > X:C and A:T congruent with H:T > O:T > X:T. These results suggested that NO-induced lesions and their base-pairs could participate in the interaction mechanisms of the DNA-binding/recognizing enzymes in a similar manner as natural nucleobases.

PHS1 regulates meiotic recombination and homologous chromosome pairing by controlling the transport of RAD50 to the nucleus

Recombination and pairing of homologous chromosomes are critical for bivalent formation in meiotic prophase. In many organisms, including yeast, mammals, and plants, pairing and recombination are intimately interconnected. The POOR HOMOLOGOUS SYNAPSIS1 (PHS1) gene acts in coordination of chromosome pairing and early recombination steps in plants, ensuring pairing fidelity and proper repair of meiotic DNA double-strand-breaks. In phs1 mutants, chromosomes exhibit early recombination defects and frequently associate with non-homologous partners, instead of pairing with their proper homologs. Here, we show that the product of the PHS1 gene is a cytoplasmic protein that functions by controlling transport of RAD50 from cytoplasm to the nucleus. RAD50 is a component of the MRN protein complex that processes meiotic double-strand-breaks to produce single-stranded DNA ends, which act in the homology search and recombination. We demonstrate that PHS1 plays the same role in homologous pairing in both Arabidopsis and maize, whose genomes differ dramatically in size and repetitive element content. This suggests that PHS1 affects pairing of the gene-rich fraction of the genome rather than preventing pairing between repetitive DNA elements. We propose that PHS1 is part of a system that regulates the progression of meiotic prophase by controlling entry of meiotic proteins into the nucleus. We also document that in phs1 mutants in Arabidopsis, centromeres interact before pairing commences along chromosome arms. Centromere coupling was previously observed in yeast and polyploid wheat while our data suggest that it may be a more common feature of meiosis.

Rad1, rad10 and rad52 Mutations Reduce the Increase of Microhomology Length during Radiation-Induced Microhomology-Mediated Illegitimate Recombination in Saccharomyces cerevisiae

Abstract Illegitimate recombination can repair DNA double-strand breaks in one of two ways, either without sequence homology or by using a few base pairs of homology at the junctions. The second process is known as microhomology-mediated recombination. Previous studies showed that ionizing radiation and restriction enzymes increase the frequency of microhomology-mediated recombination in trans during rejoining of unirradiated plasmids or during integration of plasmids into the genome. Here we show that radiation-induced microhomology-mediated recombination is reduced by deletion of RAD52, RAD1 and RAD10 but is not affected by deletion of RAD51 and RAD2. The rad52 mutant did not change the frequency of radiation-induced microhomology-mediated recombination but rather reduced the length of microhomology required to undergo repair during radiation-induced recombination. The rad1 and rad10 mutants exhibited a smaller increase in the frequency of radiation-induced microhomology-mediated recombination, and the radiation-induced integration junctions from these mutants did not show more than 4 bp of microhomology. These results suggest that Rad52 facilitates annealing of short homologous sequences during integration and that Rad1/Rad10 endonuclease mediates removal of the displaced 3' single-stranded DNA ends after base-pairing of microhomology sequences, when more than 4 bp of microhomology are used. Taken together, these results suggest that radiation-induced microhomology-mediated recombination is under the same genetic control as the single-strand annealing apparatus that requires the RAD52, RAD1 and RAD10 genes.

A Biochemically Defined System for Coding Joint Formation in V(D)J Recombination

V(D)J recombination is one of the most complex DNA transactions in biology. The RAG complex makes double-stranded breaks adjacent to signal sequences and creates hairpin coding ends. Here, we find that the kinase activity of the Artemis:DNA-PKcs complex can be activated by hairpin DNA ends in cis, thereby allowing the hairpins to be nicked and then to undergo processing and joining by nonhomologous DNA end joining. Based on these insights, we have reconstituted many aspects of the antigen receptor diversification of V(D)J recombination by using 13 highly purified polypeptides, thereby permitting variable domain exon assembly by using this fully defined system in accord with the 12/23 rule for this process. The features of the recombination sites created by this system include all of the features observed in vivo (nucleolytic resection, P nucleotides, and N nucleotide addition), indicating that most, if not all, of the end modification enzymes have been identified.

Apparent Directional Scanning for DNA Repair

Recently it was observed that the DNA repair protein human O 6-alkylguanine-DNA alkyltransferase repairs lesions at the 5′ ends of 70-nucleotide single-stranded DNA roughly threefold more frequently than lesions at the 3′ ends. Here, we introduce a coarse-grained model to show how a local asymmetry in binding kinetics (rather than thermodynamics) together with irreversible alkyl transfer can give rise to this apparent bias in sequence scanning. Exploration of the parameter space provides quantitative relationships that can be used to validate the proposed mechanism by gel-based assays.

How Directional Translocation is Regulated in a DNA Helicase Motor

PcrA helicase from Bacillus stearothermophilus is one of the smallest motor proteins structurally known in full atomic detail. It translocates progressively from the 3′ end to the 5′ end of single-stranded DNA utilizing the free energy from ATP hydrolysis. The similarities in structure and reaction pathway between PcrA helicase and F1-ATPase suggest a similar mechanochemical mechanism at work in both systems. Previous studies of PcrA translocation demonstrated a domain stepping mechanism in which, during one ATP hydrolysis cycle, the pulling together and pushing apart of two translocation domains is synchronized with alternating mobilities of the individual domains such that PcrA moves unidirectionally along single-stranded DNA. To substantiate this translocation mechanism, this study applies molecular dynamics simulations, elastic network theory, and multiple sequence alignment to analyze the system. The analysis provides further evidence that directional translocation of PcrA is regulated allosterically through synchronization of ATP hydrolysis and domain mobilities. We identify a set of essential residues coevolutionarily coupled in related helicases that should be involved in the allosteric regulation of these motor proteins.

Stimulation of fission yeast and mouse Hop2-Mnd1 of the Dmc1 and Rad51 recombinases

Genetic analysis of fission yeast suggests a role for the spHop2–Mnd1 proteins in the Rad51 and Dmc1-dependent meiotic recombination pathways. In order to gain biochemical insights into this process, we purified Schizosaccharomyces pombe Hop2-Mnd1 to homogeneity. spHop2 and spMnd1 interact by co-immunoprecipitation and two-hybrid analysis. Electron microscopy reveals that S. pombe Hop2–Mnd1 binds single-strand DNA ends of 3′-tailed DNA. Interestingly, spHop2-Mnd1 promotes the renaturation of complementary single-strand DNA and catalyses strand exchange reactions with short oligonucleotides. Importantly, we show that spHop2-Mnd1 stimulates spDmc1-dependent strand exchange and strand invasion. Ca2+ alleviate the requirement for the order of addition of the proteins on DNA. We also demonstrate that while spHop2-Mnd1 affects spDmc1 specifically, mHop2 or mHop2-Mnd1 stimulates both the hRad51 and hDmc1 recombinases in strand exchange assays. Thus, our results suggest a crucial role for S. pombe and mouse Hop2-Mnd1 in homologous pairing and strand exchange and reveal evolutionary divergence in their specificity for the Dmc1 and Rad51 recombinases.

Extensive ssDNA end formation at DNA double-strand breaks in non-homologous end-joining deficient cells during the S phase

BackgroundEfficient and correct repair of DNA damage, especially DNA double-strand breaks, is critical for cellular survival. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks in mammalian cells. The ability of other repair pathways, such as homologous recombination, to compensate for loss of NHEJ and the ways in which contributions of different pathways are regulated are far from fully understood.ResultsIn this report we demonstrate that long single-stranded DNA (ssDNA) ends are formed at radiation-induced DNA double-strand breaks in NHEJ deficient cells. At repair times ≥ 1 h, processing of unrejoined DNA double-strand breaks generated extensive ssDNA at the DNA ends in cells lacking the NHEJ protein complexes DNA-dependent protein kinase (DNA-PK) or DNA Ligase IV/XRCC4. The ssDNA formation was cell cycle dependent, since no ssDNA ends were observed in G1-synchronized NHEJ deficient cells. Furthermore, in wild type cells irradiated in the presence of DNA-PKcs (catalytic subunit of DNA-PK) inhibitors, or in DNA-PKcs deficient cells complemented with DNA-PKcs mutated in six autophosphorylation sites (ABCDE), no ssDNA was formed. The ssDNA generation also greatly influences DNA double-strand break quantification by pulsed-field gel electrophoresis, resulting in overestimation of the DNA double-strand break repair capability in NHEJ deficient cells when standard protocols for preparing naked DNA (i. e., lysis at 50°C) are used.ConclusionWe provide evidence that DNA Ligase IV/XRCC4 recruitment by DNA-PK to DNA double-strand breaks prevents the formation of long ssDNA ends at double-strand breaks during the S phase, indicating that NHEJ components may downregulate an alternative repair process where ssDNA ends are required.

On the Mechanism of Gene Amplification Induced under Stress in Escherichia coli

Gene amplification is a collection of processes whereby a DNA segment is reiterated to multiple copies per genome. It is important in carcinogenesis and resistance to chemotherapeutic agents, and can underlie adaptive evolution via increased expression of an amplified gene, evolution of new gene functions, and genome evolution. Though first described in the model organism Escherichia coli in the early 1960s, only scant information on the mechanism(s) of amplification in this system has been obtained, and many models for mechanism(s) were possible. More recently, some gene amplifications in E. coli were shown to be stress-inducible and to confer a selective advantage to cells under stress (adaptive amplifications), potentially accelerating evolution specifically when cells are poorly adapted to their environment. We focus on stress-induced amplification in E. coli and report several findings that indicate a novel molecular mechanism, and we suggest that most amplifications might be stress-induced, not spontaneous. First, as often hypothesized, but not shown previously, certain proteins used for DNA double-strand-break repair and homologous recombination are required for amplification. Second, in contrast with previous models in which homologous recombination between repeated sequences caused duplications that lead to amplification, the amplified DNAs are present in situ as tandem, direct repeats of 7–32 kilobases bordered by only 4 to 15 base pairs of G-rich homology, indicating an initial non-homologous recombination event. Sequences at the rearrangement junctions suggest nonhomologous recombination mechanisms that occur via template switching during DNA replication, but unlike previously described template switching events, these must occur over long distances. Third, we provide evidence that 3′-single-strand DNA ends are intermediates in the process, supporting a template-switching mechanism. Fourth, we provide evidence that lagging-strand templates are involved. Finally, we propose a novel, long-distance template-switching model for the mechanism of adaptive amplification that suggests how stress induces the amplifications. We outline its possible applicability to amplification in humans and other organisms and circumstances.

BRCA1 Regulates RAD51 Function in Response to DNA Damage and Suppresses Spontaneous Sister Chromatid Replication Slippage: Implications for Sister Chromatid Cohesion, Genome Stability, and Carcinogenesis

The breast/ovarian cancer susceptibility proteins BRCA1 and BRCA2 maintain genome stability, at least in part, through a functional role in DNA damage repair. They both colocalize with RAD51 at sites of DNA damage/replication and activate RAD51-mediated homologous recombination repair of DNA double-strand breaks (DSB). Whereas BRCA2 interacts directly with and regulates RAD51, the role of BRCA1 in this process is unclear. However, BRCA1 may regulate RAD51 in response to DNA damage or through its ability to interact with and regulate MRE11/RAD50/NBS1 (MRN) during the processing of DSBs into single-strand DNA (ssDNA) ends, prerequisite substrates for RAD51, or both. To test these hypotheses, we measured the effect of BRCA1 on the competition between RAD51-mediated homologous recombination (gene conversion and crossover) versus RAD51-independent homologous recombination [single-strand annealing (SSA)] for ssDNA at a site-specific chromosomal DSB within a DNA repeat, a substrate for both homologous recombination pathways. Expression of wild-type BRCA1 in BRCA1-deficient human recombination reporter cell lines promoted both gene conversion and SSA but greatly enhanced gene conversion. In addition, BRCA1 also suppressed both spontaneous gene conversion and deletion events, which can arise from either crossover or sister chromatid replication slippage (SCRS), a RAD51-independent process. BRCA1 does not seem to block crossover. From these results, we conclude that (a) BRCA1 regulates RAD51 function in response to the type of DNA damage and (b) BRCA1 suppresses SCRS, suggesting a role for this protein in sister chromatid cohesion/alignment. Loss of such control in response to estrogen-induced DNA damage after BRCA1 inactivation may be a key initial event that triggers genome instability and carcinogenesis.

An exonuclease I‐sensitive DNA repair pathway in Deinococcus radiodurans: a major determinant of radiation resistance

Deinococcus radiodurans R1 recovering from acute dose of gamma radiation shows a biphasic mechanism of DNA double-strand break repair. The possible involvement of microsequence homology-dependent, or non-homologous end joining type mechanisms during initial period followed by RecA-dependent homologous recombination pathways has been suggested for the reconstruction of complete genomes in this microbe. We have exploited the known roles of exonuclease I in DNA recombination to elucidate the nature of recombination involved in DNA double-strand break repair during post-irradiation recovery of D. radiodurans. Transgenic Deinococcus cells expressing exonuclease I functions of Escherichia coli showed significant reduction in gamma radiation radioresistance, while the resistance to far-UV and hydrogen peroxide remained unaffected. The overexpression of E. coli exonuclease I in Deinococcus inhibited DNA double-strand break repair. Such cells exhibited normal post-irradiation expression kinetics of RecA, PprA and single-stranded DNA-binding proteins but lacked the divalent cation manganese [(Mn(II)]-dependent protection from gamma radiation. The results strongly suggest that 3' (rho) 5' single-stranded DNA ends constitute an important component in recombination pathway involved in DNA double-strand break repair and that absence of sbcB from deinococcal genome may significantly aid its extreme radioresistance phenotype.

Template‐independent ligation of single‐stranded DNA by T4 DNA ligase

T4 DNA ligase is one of the workhorses of molecular biology and used in various biotechnological applications. Here we report that this ligase, unlike Escherichia coli DNA ligase, Taq DNA ligase and Ampligase, is able to join the ends of single-stranded DNA in the absence of any duplex DNA structure at the ligation site. Such nontemplated ligation of DNA oligomers catalyzed by T4 DNA ligase occurs with a very low yield, as assessed by quantitative competitive PCR, between 10(-6) and 10(-4) at oligonucleotide concentrations in the range 0.1-10 nm, and thus is insignificant in many molecular biological applications of T4 DNA ligase. However, this side reaction may be of paramount importance for diagnostic detection methods that rely on template-dependent or target-dependent DNA probe ligation in combination with amplification techniques, such as PCR or rolling-circle amplification, because it can lead to nonspecific background signals or false positives. Comparison of ligation yields obtained with substrates differing in their strandedness at the terminal segments involved in ligation shows that an acceptor duplex DNA segment bearing a 3'-hydroxy end, but lacking a 5'-phosphate end, is sufficient to play a role as a cofactor in blunt-end ligation.

Specific inhibition of the E.coli RecBCD enzyme by Chi sequences in single-stranded oligodeoxyribonucleotides.

RecBCD is an ATP-dependent helicase and exonuclease which generates 3' single-stranded DNA (ssDNA) ends used by RecA for homologous recombination. The exonuclease activity is altered when RecBCD encounters a Chi sequence (5'-GCTGGTGG-3') in double-stranded DNA (ds DNA), an event critical to the generation of the 3'-ssDNA. This study tests the effect of ssDNA oligonucleotides having a Chi sequence (Ch+) or a single base change that abolishes the Chi sequence (Chi(o)), on the enzymatic activities of RecBCD. Our results show that a 14 and a 20mer with Chi+ in the center of the molecule inhibit the exonuclease and helicase activities of RecBCD to a greater extent than the corresponding Chi(o) oligonucleotides. Oligonucleotides with the Chi sequence at one end, or the Chi sequence alone in an 8mer, failed to show Chi-specific inhibition of RecBCD. Thus, Chi recognition requires that Chi be flanked by DNA at either end. Further experiments indicated that the oligonucleotides inhibit RecBCD from binding to its dsDNA substrate. These results suggest that a specific site for Chi recognition exists on RecBCD, which binds Chi with greater affinity than a non-Chi sequence and is probably adjacent to non-specific DNA binding sites.

Functional characterization of the interacting domains of the positive coactivator PC4 with the transcription factor AP-2α

The transcriptional positive cofactor 4 (PC4) physically interacts with the transcription factor, activator protein-2 (AP-2) α, and overexpression of PC4 results in a relief of the AP-2 transcriptional self-interference, which is induced by high levels of AP-2α expression. PC4 was initially described as a DNA-binding protein that enhances the activator-dependent transcription of class II genes in vitro, but it was later shown that PC4 could also act as a potent repressor of transcription on specific DNA structures such as single-stranded (ss) DNA, DNA ends and heteroduplex DNA. To further explore the functional domains of PC4 and its ssDNA-binding effect in the interaction with AP-2α and on AP-2 transcriptional activity, we investigated the C-terminal domain of PC4 (PC4-CTD) and several PC4 mutants in which the ssDNA binding function was interrupted. We found that the C-terminal domain of PC4 physically interacts with AP-2α and retains the function of full-length protein in relieving transcription self-interference of AP-2. A point-mutated form of PC4 within the C-terminal domain β-ridge, PC4 W89A, or a triple mutant in the β2–β3 loop of PC4, F77A/K78G/K80G, inactivate the ability of PC4 to bind AP-2α and to relieve the transcription self-interference of AP-2α. In addition, point-mutated forms of AP-2α within the activation domain (AD) that inactivate AP-2 transcription activity also lose their self-interference function. Our data suggest that the C-terminal domain of the transcription cofactor PC4 is critical for AP-2α transcriptional interference that is mediated by the activation domain of AP-2α.

Translocation and gross deletion breakpoints in human inherited disease and cancer II: Potential involvement of repetitive sequence elements in secondary structure formation between DNA ends.

Translocations and gross deletions are responsible for a significant proportion of both cancer and inherited disease. Although such gene rearrangements are nonuniformly distributed in the human genome, the underlying mutational mechanisms remain unclear. We have studied the potential involvement of various types of repetitive sequence elements in the formation of secondary structure intermediates between the single-stranded DNA ends that recombine during rearrangements. Complexity analysis was used to assess the potential of these ends to form secondary structures, the maximum decrease in complexity consequent to a gross rearrangement being used as an indicator of the type of repeat and the specific DNA ends involved. A total of 175 pairs of deletion/translocation breakpoint junction sequences available from the Gross Rearrangement Breakpoint Database [GRaBD; www.uwcm.ac.uk/uwcm/mg/grabd/grabd.html] were analyzed. Potential secondary structure was noted between the 5' flanking sequence of the first breakpoint and the 3' flanking sequence of the second breakpoint in 49% of rearrangements and between the 5' flanking sequence of the second breakpoint and the 3' flanking sequence of the first breakpoint in 36% of rearrangements. Inverted repeats, inversions of inverted repeats, and symmetric elements were found in association with gross rearrangements at approximately the same frequency. However, inverted repeats and inversions of inverted repeats accounted for the vast majority (83%) of deletions plus small insertions, symmetric elements for one-half of all antigen receptor-mediated translocations, while direct repeats appear only to be involved in mediating simple deletions. These findings extend our understanding of illegitimate recombination by highlighting the importance of secondary structure formation between single-stranded DNA ends at breakpoint junctions.