Báo cáo khoa học: Effect of monovalent cations and G-quadruplex structures on the outcome of intramolecular homologous recombination doc

Effect of monovalent cations and G-quadruplex structures on the outcome of intramolecular homologous recombination ´ ´ ´ Paula Barros*, Francisco Boan*, Miguel G Blanco and Jaime Gomez-Marquez ´ ´ ´ Departamento de Bioquımica e Bioloxıa Molecular, Facultade de Bioloxıa-CIBUS, Universidade de Santiago de Compostela, Spain Keywords G-quadruplex; minisatellite MsH43; monovalent cations; recombination fidelity; repetitive sequences Correspondence ´ ´ J Gomez-Marquez, Departamento de ´ ´ Bioquımica e Bioloxıa Molecular, Facultade ´ de Bioloxıa-CIBUS, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Spain Fax: +34 9815969054 Tel: +34 981563100 (ext 16937) E-mail: jaime.gomez.marquez@usc.es *These authors contributed equally to this work (Received 17 February 2009, revised 18 March 2009, accepted 20 March 2009) doi:10.1111/j.1742-4658.2009.07013.x Homologous recombination is a very important cellular process, as it provides a major pathway for the repair of DNA double-strand breaks This complex process is affected by many factors within cells Here, we have studied the effect of monovalent cations (K+, Na+, and NH4+) on the outcome of recombination events, as their presence affects the biochemical activities of the proteins involved in recombination as well as the structure of DNA For this purpose, we used an in vitro recombination system that includes a protein nuclear extract, as a source of recombination machinery, and two plasmids as substrates for intramolecular homologous recombination, each with two copies of different alleles of the human minisatellite MsH43 We found that the presence of monovalent cations induced a decrease in the recombination frequency, accompanied by an increase in the fidelity of the recombination Moreover, there is an emerging consensus that secondary structures of DNA have the potential to induce genomic instability Therefore, we analyzed the effect of the sequences capable of forming G-quadruplex on the production of recombinant molecules, taking advantage of the capacity of some MsH43 alleles to generate these kinds of structure in the presence of K+ We observed that the MsH43 recombinants containing duplications, generated in the presence of K+, did not include the repeats located towards the 5¢-side of the G-quadruplex motif, suggesting that this structure may be involved in the recombination events leading to duplications Our results provide new insights into the molecular mechanisms underlying the recombination of repetitive sequences The integrity of chromosomal material is dependent upon the efficient repair of DNA double-strand breaks (DSBs), which arise during DNA replication or are caused by exogenous agents Without such systems, unrepaired breaks can lead to chromosomal translocations, loss of transcriptional control, and promotion of tumorigenesis [1] In human cells, the repair of DSBs can take place through two independent systems: homologous recombination (HR), and nonhomologous end-joining HR is promoted by several enzymes of the RAD52 epistasis group, which includes RAD51, the human homolog of Escherichia coli RecA [2,3] This protein promotes the key homologous pairing and strand-exchange reactions leading to the formation of interlinked recombination intermediates [4] Changes in ionic strength alter the behavior of some enzymes involved in the HR process In this regard, previous work has shown that high salt concentrations provoke conformational changes in the RAD51 protein [5] favoring the coaggregation of RAD51–ssDNA nucleoprotein filaments with duplex DNA, stimulating the recombination [6] More recently, a study defining the effect of salt on human RAD51 activities was reported [7] However, as far as we know, none of the Abbreviations DSB, double-strand break; HR, homologous recombination FEBS Journal 276 (2009) 2983–2993 ª 2009 The Authors Journal compilation ª 2009 FEBS 2983 Effect of cations and G-quartets on recombination P Barros et al studies on the influence of salts on the recombination process have analyzed how monovalent cations could affect the fidelity of recombination, defined as the percentages of equal and unequal recombinant molecules, and the frequency of recombination In our laboratory, we have developed an in vitro system with which to analyze HR and nonhomologous end-joining [8–11] This system allows us to establish in vitro the recombinogenic capacity of any DNA sequence, as well as to determine the nature of the recombinant molecules generated In the present study, we employed this in vitro system to analyze the effect of the monovalent cations K+, Na+ and NH4+ on recombination For this purpose, we used the minisatellite MsH43, a human DNA sequence composed of pentamers and hexamers organized in a tandem array [12,13] The organization in tandem of small repeat units provides a good substrate with which to study the frequency of equal and unequal crossovers, as it facilitates perfect and nonperfect pairings We found that the presence of monovalent cations led to a higher proportion of equal recombinants, and hence an increase in the fidelity of the recombination events in the experimental system employed On the other hand, it is well known that guaninerich nucleic acids (DNA and RNA) are capable of forming four-stranded structures named G-quadruplexes (also known as G-tetrads, G4s, or G-quartets) [14,15] These structures are further stabilized by the presence of a monovalent cation (especially K+) in the center of the tetrad [15,16] G-quadruplex-forming sequences have been identified in eukaryotic telomers, as well as in gene promoters, recombination sites, and DNA tandem repeats [15] Whether or not genomic G-rich structures can form quadruplex-based structures in vivo remains to be fully demonstrated, although supportive data are starting to emerge [14,15,17,18] G-quadruplexes have long been hypothesized to play roles in DNA recombination Thus, G-quadruplex DNA might play a role in class switch recombination in the immunoglobulin genes [19,20], and studies in yeast suggest possible roles for G-quadruplex DNA in homologous recombination during meiosis [21] In relation to this, Hop1 not only binds to and catalyzes the formation G-quadruplex DNA in vitro, but also promotes the pairing of dsDNA molecules via quadruplex structures [22] However, is not yet clear how the in vitro activities of this and other proteins on G-quadruplex DNA relate to their in vivo functions In the present work, we also analyzed the effect of the presence of sequences capable of forming G-quadruplex structures on recombination frequency and the generation of recombinant molecules, taking advantage 2984 of the capacity of the minisatellite MsH43 to form this kind of structure in the presence of K+ [12] We found that the presence of G-quadruplex did not alter the recombination frequency as compared with the allele control Moreover, the great majority of recombinants containing duplications generated in the presence of K+ did not include the repeats located at the 5¢-side of the G-quadruplex motif of MsH43, suggesting that this structure is involved in the recombination events leading to duplications A model to explain this finding, involving replication slippage, is also shown Results and discussion To study the effects of salts on the frequencies of equal and unequal recombinant products generated in the recombination experiments, we employed the minisatellite MsH43, as it shows two useful features: (a) an organization in tandem, which allows the existence of different types of homologous pairings (in register or not in register), leading to the formation of equal and unequal recombinant molecules; and (b) the ability of allele 80.1 to form a G-quadruplex, as it contains the motif (TGGGGC)4, which is a G-quadruplex-forming structure, and the inability of allele 73.1 to form such a structure, because it contains the motif TGGGGC repeated only three times instead of four [12]; this differential characteristic allows analysis of the effect of the presence of G-quadruplex structures on the generation of recombinant molecules in the in vitro system employed in this work To carry out the recombination analyses, we employed an in vitro system designed to detect intramolecular homologous recombination events [8–11] We constructed two pBR322-based plasmids, the recombinant substrates p73.1 and p80.1, bearing two copies cloned in the same orientation as the corresponding MsH43 allele, 73.1 or 80.1 The map of these recombinant substrates is shown in Fig 1A As the lacZ gene is situated between the two copies of the MsH43 inserts, the recombination that takes place after pairing of the MsH43 homologous sequences leads to the excision of the lacZ gene from the original substrate, generating two kind of recombinants, equal and unequal (Fig 1B) In the equal recombinants, the minisatellite remains unaltered, whereas in the unequal ones, the minisatellite displays size variations caused by unequal pairings or alterations during the recombination process In the recombination experiments, each plasmid substrate was incubated with the nuclear extract under standard conditions [8] After incubation, DNA was extracted and used to transform bacteria The recombi- FEBS Journal 276 (2009) 2983–2993 ª 2009 The Authors Journal compilation ª 2009 FEBS P Barros et al Effect of cations and G-quartets on recombination Recombination substrates A Sc E P02.1 P02.2 E S E P02.1 E E MsH43 73.1 MsH43 80.1 p73.1 p80.1 ori ori MsH43 73.1 E B MsH43 MsH43 80.1 E Pairing of homologous sequences Rec substrate E LacZ Crossover MsH43 Recombinant product (LacZ–) X ori MsH43 Fig Map of the recombination substrates and representation of an intramolecular homologous recombination event (A) Plasmids p73.1 and p80.1 (not drawn to scale) were used as substrates in the recombination assays They contain a replication origin (ori), an ampicillin resistance gene (Amp), the lacZ gene, and two identical copies of the MsH43 sequence (wide black arrows), cloned in the same orientation At the top is shown a scheme of the MsH43 inserts, minisatellite (open box), and flanking sequences; thin arrows mark the position of primers P02.1 and P02.2 E, EcoRI; Sc, SacII; S, SalI (B) The intramolecular homologous recombination generates two kinds of plasmid: equals, in which the MsH43 remains unaltered, and unequals, with alterations in the minisatellite sequence P02.2 E MsH43 80.1 MsH43 73.1 nant plasmids generated lacZ) bacteria (white colonies), and the original plasmids generated lacZ+ bacteria (blue colonies) The recombinant plasmids were first analyzed by restriction with EcoRI The digestion of p73.1 recombinant products yielded two restriction fragments, one with the minisatellite sequence, and the digestion of the p73.1 original plasmid generated five restriction fragments, two of them identical (those containing the minisatellite) (Fig 2A) If the recombinant was unequal, then the EcoRI fragment containing the minisatellite showed size variations (asterisks in Fig 2A) In the case of the p80.1, the digestion with EcoRI of the original plasmid yielded four DNA fragments, whereas the digestion of its recombinant products generated only one fragment (Fig 2B) To facilitate the identification of p80.1 recombinants, the recombinant plasmids were amplified by PCR with the primers P02.1 and P02.2 [12] The analysis of amplifications products allowed differentiation of the majority of the equal and unequal events (Fig 2C) However, when the variation affected few repeats, it was difficult to distinguish length variations of the minisatellite To solve this problem, heteroduplex analyses [13] were carried out by mixing the amplification products of each recombinant with the PCR product obtained from the MsH43 sequence present in the original construct Figure 2D shows the result of a heteroduplex assay employing the recombinants generated in the experiments with p80.1 The generation of heteroduplex molecules denotes the presence of an unequal Amp ori Equal recombinant ori Unequal recombinant recombinant, whereas the absence of heteroduplex molecules means that it is an equal recombinant The sequencing of 14 equal recombinants corroborated that the recombinant molecules classified as equal conserved the original minisatellite sequence (data not shown) The results of the intramolecular homologous recombination experiments are summarized in Table The data were collected from three independent experiments for each assay condition (standard or supplemented with salts) The LacZ+ colonies were produced by the transformation with the original plasmids, whereas the LacZ) colonies were the result of transforming bacteria with the recombinant plasmids The lacZ) colonies due to mutations in the lacZ gene made up less than 1% of the total lacZ) colonies, and the frequency of the lacZ) colonies in transformations with the original substrate plasmids not exposed to the nuclear extract or with heat-inactivated extract (15 min, 100 °C) was about · 10)5 Under standard conditions, both the recombination frequencies and the frequency of equal and unequal recombinants were very similar with plasmids p73.1 and p80.1 Noteworthy, with both plasmids, the recombination events that generated unequal recombinants were more abundant ( 20%) than those that maintained the original sequence of MsH43 To verify the effect of the presence of G-quadruplex-forming sequences in this in vitro system, we performed several assays in the presence of 20 mm K+, added to stabi- FEBS Journal 276 (2009) 2983–2993 ª 2009 The Authors Journal compilation ª 2009 FEBS 2985 p80.1 7 10 11 ** * * D M p80.1 * * C 10 11 12 13 14 15 * * P Barros et al * * * * * B 8 p80.1 A p73.1 Effect of cations and G-quartets on recombination 12 13 14 15 16 17 18 19 20 21 22 23 * * ** * * * 10 11 12 13 14 15 16 17 18 Heteroduplex molecules lize the G-quadruplex structure Once again, the recombination frequencies were very similar with both plasmids, indicating that the capacity of the MsH43 2986 Fig Analysis of the recombinant products generated in the in vitro recombination assays (A) Analysis in 1.5% agarose gels of the EcoRI digest of the recombinant plasmids obtained from lacZ) colonies in experiments carried out with p73.1 The EcoRI restriction pattern of the original plasmid p73.1 (lane p73.1) and the recombinant products (lanes 1–15) are shown The arrow indicates the DNA fragment that contains the original MsH43 sequence, and asterisks mark the DNA fragment containing alterations in the size of MsH43 (unequal recombinants) (B) Analysis in 1.5% agarose gels of the EcoRI digest of the plasmids obtained from lacZ) colonies in recombination experiments carried out with p80.1 The EcoRI restriction patterns corresponding to the original plasmid p80.1 (lane p80.1) and the recombinant products (lanes 1–9) are shown (C) Analysis in 2% agarose gels of the amplification products of recombinant plasmids obtained in experiments carried out with p80.1 The amplification product of the original plasmid p80.1 (lane p80.1) and the recombinant products (lanes 1–23) are shown The arrow indicates the DNA fragment that contains the original MsH43 sequence, and asterisks mark unequal recombinants (D) Heteroduplex analysis in a 5% polyacrylamide gel (the presence of heteroduplex molecules denotes the presence of an unequal recombinant); lane p80.1 is shown as a control of no heteroduplex formation The arrow indicates the DNA fragment that contains the original MsH43 sequence M, 100 bp ladder (Promega) 80.1 allele to form G-quadruplex does not influence the recombination frequency, at least in our in vitro system However, the presence of K+ caused a strong FEBS Journal 276 (2009) 2983–2993 ª 2009 The Authors Journal compilation ª 2009 FEBS P Barros et al Effect of cations and G-quartets on recombination Table Quantitative analysis of recombination experiments The recombinant frequencies are given as the ratio between the number of LacZ) colonies and the total number of colonies obtained in each assay The equal and unequal recombinant frequencies are given as the ratio between the total number of each type of recombinant and the total number of recombinants For columns 4, and 8, mean ± standard deviation of the data is provided Equal recombinants Recombination substrate Assay conditions Colonies (LacZ+ ⁄ LacZ)) p73.1 Standard 3863 ⁄ 42 4034 ⁄ 45 4060 ⁄ 50 p80.1 Standard 7832 ⁄ 80 6095 ⁄ 68 6866 ⁄ 74 p73.1 20 mM KCl 8296 ⁄ 39 9183 ⁄ 47 7850 ⁄ 35 p80.1 20 mM KCl 9450 ⁄ 39 12 365 ⁄ 47 9617 ⁄ 44 p80.1 20 mM NaCl 19 559 ⁄ 29 17 360 ⁄ 27 16 261 ⁄ 21 p80.1 20 mM NH4Cl 8114 ⁄ 23 8555 ⁄ 25 7962 ⁄ 22 1.087 1.116 1.232 1.145 1.021 1.116 1.078 1.072 0.470 0.512 0.448 0.476 0.413 0.380 0.458 0.417 0.148 0.156 0.129 0.144 0.283 0.292 0.276 0.284 reduction (near 50%) in the recombination frequencies, as well as an important decrease in the nuclease activity of the nuclear extract (data not shown) As the initiation of homologous recombination is mediated by a nuclease activity that introduces DNA DSBs [23], it is possible that the reduction in recombination frequency was due to inhibition of the nuclease activity by K+ Remarkably, in the presence of K+, the proportions of equal and unequal recombinants were inverted with respect to the results obtained under standard conditions; that is, the equal recombinants were more abundant ( 25%) than the unequal ones (Table 1) Was this inversion produced specifically by K+? The recombination assays carried out in the presence of Na+ or NH4+, maintaining the same ionic strength, showed a marked reduction of the recombination frequency with respect to the standard conditions, more pronounced than with K+, and also a predominance of equal recombinants (Table 1) The finding that Na+ and NH4+ caused a greater decrease in the nuclease activity of the nuclear extract than K+ (data not shown) provides a coherent explanation of the observation that the Unequal recombinants No Frequency (%) No Frequency (%) 16 21 19 Recombination frequency (%) 0.414 0.521 0.468 0.468 0.447 0.459 0.422 0.442 0.289 0.305 0.293 0.296 0.265 0.283 0.322 0.290 0.118 0.121 0.098 0.112 0.222 0.245 0.314 0.260 26 24 31 0.673 0.595 0.764 0.677 0.574 0.657 0.656 0.629 0.181 0.207 0.155 0.181 0.148 0.097 0.136 0.127 0.030 0.035 0.031 0.032 0.061 0.047 0.062 0.057 ± 0.077 35 28 29 ± 0.048 24 28 23 ± 0.033 25 35 31 ± 0.039 23 21 16 ± 0.014 18 21 17 ± 0.008 ± 0.054 45 40 45 ± 0.018 15 19 12 ± 0.008 14 12 13 ± 0.029 6 ± 0.013 5 ± 0.048 ± 0.085 ± 0.048 ± 0.026 ± 0.027 ± 0.003 ± 0.008 recombination frequencies obtained with those cations were lower than with K+ Cations play essential roles in nucleic acid and protein structure, stability, folding, and catalysis By means of their interactions with DNA and proteins, they could play an important role in recombination For instance, changes in K+ concentration could alter chromatin structure by taking advantage of the unique sensitivity of quadruplex formation to K+ and other cations present in the cells [24,25] On the other hand, in vitro studies with G-rich telomeric DNA sequences and the minisatellite MsH43 have shown that they can form quadruplex structures whose stability is sensitive to changes in the concentrations of important physiological cations such K+ [12,16] As mentioned earlier, RAD51, a key enzyme in HR, is affected by salts Our results provide strong evidence that the presence of monovalent cations causes a strong decrease in recombination frequency, probably due to inhibition of the nuclease activity that produces DSBs on the plasmid substrates, and leads to enhancement of the fidelity of recombination, as the proportion of equal recombinants was higher The presence of monovalent cations FEBS Journal 276 (2009) 2983–2993 ª 2009 The Authors Journal compilation ª 2009 FEBS 2987 Effect of cations and G-quartets on recombination P Barros et al in the nucleus of the cells is an important physiological requirement, and our results suggest that monovalent cations also influence genomic stability through their participation in the recombination process This increase in fidelity could be related to alterations in the structure of the minisatellite and to conformational changes in proteins involved in recombination In this regard, it has been reported that high salt induces conformational changes in RAD51, leading to the formation of interlinked recombination intermediates [2] that are essential for the correct progression of the recombination process It is worth noting that, with the in vitro system developed in our laboratory, we did not observe an influence of the capacity to generate G-quadruplex DNA on the recombinogenic frequency of the minisatellite MsH43 A The existence of unequal recombinants allowed a search for the sites where the rearrangement in the MsH43 occurred The analysis of 163 recombinant sequences (Figs and 4) revealed that duplications occurred less frequently (30%) than deletions, similar to what was reported for the minisatellite CEB1 [26,27], suggesting that this type of repetitive sequence is prone to undergoing deletions in the recombination process Most of the unequal recombinants involve simple deletions (Fig 3), and only four recombinants derived from p80.1 (S5, S11, K10 and K13 in Fig 3A) showed double deletions, suggesting that they did not arise by a simple recombination event With regard to the MsH43 expansions, they seem to be the consequence of simple direct duplications (Fig 4), except in one case derived from p73.1, where one repeat was B Fig Organization of MsH43 recombinants containing deletions (A) Sequence array of the deleted molecules obtained in experiments with p80.1: standard conditions (S1–S18), with K+ (K1–K21), with Na+ (Na1–Na9), and with NH4+ (N1–N4); asterisks indicate recombinants with double deletions The sequence of the MsH43 80.1 allele is shown (B) Sequence array of the deleted molecules obtained in experiments with p73.1: standard conditions (S1–S17) and with K+ (K1–K19) The sequence of the MsH43 73.1 allele is shown The discontinuity in the sequence indicates the deleted fragment Minisatellite repeats are depicted by a color code shown at the bottom 2988 FEBS Journal 276 (2009) 2983–2993 ª 2009 The Authors Journal compilation ª 2009 FEBS P Barros et al A MsH43 80.1 allele S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 K1 K2 K3 K4 K5 K6 K7 K8 K9 K10 K11 K12 K13 K14 Effect of cations and G-quartets on recombination B MsH43 73.1 allele S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 K1 K2 K3 K4 K5 K6 K7 K8 K9 Na1 Na2 Na3 Na4 Na5 Na6 Na7 N1 N2 N3 N4 N5 N6 N7 K10 K11 K12 K13 K14 K15 K16 K17 K18 K19 Fig Organization of MsH43 recombinants containing duplications (A) Sequence array of recombinants with duplications obtained in experiments with p80.1: standard conditions (S1–S13), K+ (K1–K14), Na+ (Na1–Na7), and NH4+ (N1–N7) The sequence of the MsH43 80.1 allele is shown (B) Sequence array of recombinants presenting duplications obtained in experiments with p73.1 under standard conditions (S1–S15) and with K+ (K1–K19) The sequence of the MsH43 73.1 allele is shown The marks in the recombinant S1 denote the mutations with respect to the original MsH43 allele The arrowhead indicates the recombinant that has a repeat intercalated between the duplicated arrays Arrows indicate the duplicated fragment intercalated between the duplicated fragments (S5 in Fig 4B) None of the recombinants analyzed showed truncated repeats This feature was also observed in the recombinants generated by the human minisatellite MsH42 [8,9] and by the human minisatellite CEB1 inserted in yeast [26,27], indicating that the reorganizations produced in the minisatellite MsH43 arose by a homology-guided mechanism Interestingly, in the presence of K+, the p80.1 recombinants displaying duplications did not include the repeats located at the 5¢-side of the G-quadruplex motif (K1–K14 in Fig 4A) In contrast, this limitation was not found in the assays carried out either under standard conditions or in the presence of Na+ or NH4+, or with p73.1 (Fig 4) It is tempting to speculate that the G-quadruplex motif (TGGGGC)4 influences the resolution of the recombination events, leading to duplications in the minisatellite sequence There is an emerging consensus that secondary structures of DNA have the potential to induce genomic instability The role of nonlinear DNA in replication, recombination and transcription has become evident in recent years Several studies have predicted and characterized regulatory elements at the sequence level However, little is known about the role of DNA structures as regulatory motifs Cells use these structural motifs as signals for processes such as gene regulation or recombination in both prokaryotes and eukaryotes [28,29] The coincidence of breakpoints of gross deletions with non-B DNA conformations has led to the conclusion that these structures can trigger genomic rearrangements through recombination ⁄ repair FEBS Journal 276 (2009) 2983–2993 ª 2009 The Authors Journal compilation ª 2009 FEBS 2989 Effect of cations and G-quartets on recombination P Barros et al activities [30] Furthermore, G-quadruplex secondary structures can induce genetic rearrangements and promote RecA-independent homologous recombination [31] Genome-wide predictions have shown an abundance of G-quadruplex DNA motifs in the genomes of Homo sapiens [32] and E coli [33] In both species, the distribution of G-quadruplex structures seems to be nonrandom and linked to regulatory regions of the genome The important finding is that this kind of structure may play a role in genome dynamics at three levels: regulation of transcription, recombination and mutation hotspots in vivo, and blocking the progression of DNA polymerases [34,35] Our results demonstrate that a minisatellite sequence, which is not included inside a gene [12], can form Gquadruplex structures that interfere with DNA synthesis and influence the resolution of recombination It is tempting to speculate that this type of repetitive sequence could be involved in processes related to genome stability In this regard, although the mechanisms involved in minisatellite instability are poorly understood, some relevant factors have already been found, such as the requirement for DSBs [23] and length and sequence heterozygosity [36] Furthermore, size alterations of G-rich minisatellites can be caused by the ability of these sequences to adopt G-quadruplex structures [37] and by their capacity to undergo slippage during replication or unequal crossovers [8,9,38] In the case of MsH43, we observed that the recombinants containing duplications, generated in the presence of K+, did not include the repeats located at the 5¢-side of the G-quadruplex motif of MsH43, suggesting that this structure is involved in the recombination events leading to duplications In Fig 5, we show a hypothetical model to explain the mechanism involved in the generation of duplications in the presence of a G-quadruplex According to this, the generation of duplications is explained by replication slippage on the strand of new synthesis The presence of a G-quadruplex structure stabilized by K+ in the slippage loop would interfere with the replication at the 5¢-end of the G-quadruplex motif and consequently with the generation of duplications containing this 5¢-region of MsH43 This effect is not observed if the slippage occurs either on the 3¢-side of the G-quadruplex DNA structure or in the template strand; in the latter case, the slippage would produce the observed deletions This model explains the generation of all duplications derived from the experiments with p80.1 in the presence of K+, with the exception of K1, which contains the G-quadruplex motif in the duplicated sequence (Fig 4A) Possible explanations for the generation of the recombinant K1 could be that not all p80.1 plasmid molecules present in the recombina2990 A 5´ ´ (TGGGGC)4 ´ 5´ DSB B C 5´ 3´ 5´ 5´ 3´ 5´ 3´ G4 structure 5´ Slippage loop 5´ 3´ Slippage loop 5´ Fig Recombination model involving G-quadruplex (G4) structure and slippage processes for the generation of recombinant molecules containing duplications and deletions (A) Generation of a DSB by the nuclease(s) of the nuclear extract initiates the recombination process; the sequence (TGGGGC)4 represents the G-quadruplex motif in the MsH43 80.1 allele (B) After the break, there is a strand invasion of the homologous sequence in the second copy of the minisatellite included in the recombinant plasmid employed in the assay (C) Replication slippage may occur in the strand of new synthesis (left side) If the slippage loop comprises the region containing the G-quadruplex motif, a G-quadruplex structure could be generated that would be stabilized by the presence of K+; this secondary structure would interfere with DNA synthesis, avoiding the formation of duplications involving the 5¢-side of the G-quadruplex motif When the slippage loop is located at the 3¢-side of the G-quadruplex DNA motif [right side of (C)], duplications can be generated without the interference of G-quadruplex structures According to this model, it is worth noting that the slippage in the template strand, leading to deletions in the MsH43 sequence, would not be affected by the generation of G-quartets, as it does not have the G-quadruplex DNA motif tion assay formed G-quadruplex, or that this was unstable In both cases, the slippage process would not be interfered with by the G-quadruplex structure, making possible the inclusion of the sequence (TGGGGC)4 in some recombinant molecules Supporting this reasoning are the results found in dimethyl sulfate methylation protection assays carried out with oligonucleotides designed from the sequence of several MsH43 alleles [12] In these experiments, even at concentrations of 100 mm K+, there is a residual amount of oligonucleotides that not form G-quadruplex The present work shows that the presence of monovalent cations increases the fidelity of recombination, and that this effect is independent of the presence of G-quadruplex structures in the minisatellite MsH43 However, the G-quadruplex structure seems to be a barrier to the events leading to duplications, perhaps leading to blockage of polymerases at that point Therefore, the G-quadruplex would not be a stimulus for recombination but a source of genomic instability FEBS Journal 276 (2009) 2983–2993 ª 2009 The Authors Journal compilation ª 2009 FEBS P Barros et al It should be noted that the results obtained with MsH43 cannot be applied to any DNA sequence, as the repetitive nature of MsH43 favors the existence of unequal crossovers as well as slippage processes Perhaps one of the functions of repetitive DNA in the genomes is to serve as instability spots that are necessary for genome evolution Finally, the results presented here show that the in vitro system used in this study may be useful for investigation of the mechanisms involved in recombination and DNA instability, as well for the analysis of how monovalent cations affect the proteins implicated in this fundamental biological process Experimental procedures Recombination substrates The alleles of MsH43 used in this study, 73.1 and 80.1, were obtained by amplification of human genomic DNA, with the primers P02.1 and P02.2 [10] The PCR products were cloned in the pGEM-T Easy vector (Promega, Madison, WI, USA) The plasmid containing the 73.1 allele was digested with SacII–SalI, generating a 569 bp fragment, and the plasmid containing the 80.1 allele was digested with EcoRI, producing a 586 bp fragment To generate the recombination substrates, plasmids p73.1 and p80.1, two identical copies of each fragment were cloned in pBR322, in the same orientation, flanking the lacZ gene (Fig 1A) In vitro recombination assays We have previously shown that the recombination products are generated by the nuclear extract and not by the repair machinery of bacteria [7] The standard recombination reactions were performed in a final volume of 100 lL containing 20 mm Tris ⁄ HCl (pH 7.5), 10 mm MgSO4, mm ATP, 0.1 mm each dNTP, lg of plasmid (p73.1 or p80.1), and 10 lg of rat testis nuclear extract [6] In the experiments containing KCl, NaCl, or NH4Cl, the salts were added to a final concentration of 20 mm Several concentrations of salts were assayed (5, 10, 15, 20, and 25 mm), and a concentration of 20 mm was chosen for the recombination experiments, because it produces a good number of white colonies, allowing the screening of many recombinants per assay (data not shown) Concentrations higher than 20 mm produced few white colonies, probably because of the decrease caused in the recombination frequency, which could be due to inhibition of nuclease activity The inhibition of nuclease activity was observed by the analysis of plasmid integrity, after incubation with the nuclear extract in the presence of the different monovalent cation concentrations, by electrophoresis on agarose gels After incubation of recombination substrates with the Effect of cations and G-quartets on recombination nuclear extracts for 30 at 37 °C, DNA was phenolextracted, ethanol-precipitated, and used to transform E coli DH5a cells Bacteria were plated onto LB agar plates containing Blue-O-Gal (BRL, Gaithersburg, MD, USA) at 0.3 gỈL)1 as lacZ gene indicator We observed that lacZ) colonies due to mutations in the lacZ gene made up less than 1% of the total lacZ) colonies The frequency of the lacZ) colonies in transformations with the original substrate plasmids not exposed to the nuclear extract or with heat-inactivated extract (15 min, 100 °C) was about · 10)5 The white colonies (recombinant products) were used for minipreparation of plasmid DNA and aliquots of  300 ng were digested with EcoRI, and analyzed in agarose gels PCR, DNA sequencing, and heteroduplex analysis The PCR reactions were performed in 25 lL containing PCR buffer [67 mm Tris ⁄ HCl, pH 8.8, 16 mm (NH4)2SO4, 0.01% Tween-20], 0.1 ng of plasmid, 0.3 lm each primer (P02.1 and P02.2), 0.2 mm dNTPs, 1.5 mm MgCl2, and 0.5 U of Taq polymerase Cycling conditions were 29 cycles of 95 °C for min, 56 °C for 30 s, and 72 °C for 40 s, and a final cycle with an extension of When the PCR products were used for direct cycle sequencing employing the dGTP BigDye Terminator v3.0 Sequencing kit (Applied Biosystems, Foster City, CA, USA), they were treated with exonuclease I and alkaline phosphatase (Exo ⁄ Sap-It) (USB, Cleveland, OH, USA) After this treatment, the PCR products were cycle sequenced by 25 cycles of 96 °C for 10 s and 68 °C for in a PTC-200 thermocycler (MJ Research, Ramsey, MN, USA), and purified by ethanol precipitation The sequencing products were analyzed using the 377 DNA Automated Sequencer (Applied Biosystems) For the heteroduplex analysis, aliquots of lL of the PCR products obtained from the recombinants and from the original recombination substrates were mixed at 95 °C for min, and slowly cooled to room temperature The heteroduplex molecules were detected by electrophoresis in 5% polyacrylamide gels (29 : 1) at a constant voltage of 140 V for h using 1· TBE buffer (0.09 m Tris ⁄ borate, 0.002 m EDTA) and visualized by ethidium bromide staining Acknowledgements This work was supported by the Spanish Ministerio de ´ Educacion y Ciencia (BFU2006-06708) and by the Xunta de Galicia (PGIDT07PX12001099R) 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ª 2009 The Authors Journal compilation ª 2009 FEBS 2993 .. .Effect of cations and G-quartets on recombination P Barros et al studies on the influence of salts on the recombination process have analyzed how monovalent cations could affect the fidelity of. .. involved in the generation of duplications in the presence of a G-quadruplex According to this, the generation of duplications is explained by replication slippage on the strand of new synthesis The. .. the nuclear extract in the presence of the different monovalent cation concentrations, by electrophoresis on agarose gels After incubation of recombination substrates with the Effect of cations