Effect of monovalent cations and G-quadruplex structures on the outcome of intramolecular homologous recombination Paula Barros*, Francisco Boa´ n*, Miguel G. Blanco and Jaime Go´ mez-Ma´ rquez
Departamento de Bioquı´mica e Bioloxı´a Molecular, Facultade de Bioloxı´a-CIBUS, Universidade de Santiago de Compostela, Spain
Keywords G-quadruplex; minisatellite MsH43; monovalent cations; recombination fidelity; repetitive sequences
Correspondence J. Go´ mez-Ma´ rquez, Departamento de Bioquı´mica e Bioloxı´a Molecular, Facultade de Bioloxı´a-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)
Homologous recombination is a very important cellular process, as it pro- vides a major pathway for the repair of DNA double-strand breaks. This complex process is affected by many factors within cells. Here, we have +) on the studied the effect of monovalent cations (K+, Na+, and NH4 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 recombina- tion, 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 recombi- nants 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.
protein promotes the key homologous pairing and strand-exchange reactions leading to the formation of interlinked recombination intermediates [4].
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 transloca- tions, 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
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 pro- tein [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
doi:10.1111/j.1742-4658.2009.07013.x
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Abbreviations DSB, double-strand break; HR, homologous recombination.
studies on the influence of salts on the recombination process have analyzed how monovalent cations could affect the fidelity of recombination, defined as the per- centages of equal and unequal recombinant molecules, and the frequency of recombination.
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 find- ing, involving replication slippage, is also shown.
Results and discussion
In our laboratory, we have developed an in vitro sys- tem 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 + on of the monovalent cations K+, Na+ and NH4 recombination. For this purpose, we used the minisat- ellite 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 recombinants, and hence an proportion of equal increase in the fidelity of the recombination events in the experimental system employed.
To study the effects of salts on the frequencies of equal and unequal recombinant products generated in the recombination experiments, we employed the mini- satellite 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 dif- ferential characteristic allows analysis of the effect of the presence of G-quadruplex structures on the genera- tion of recombinant molecules in the in vitro system employed in this work. the
To carry out
On the other hand, it is well known that guanine- rich nucleic acids (DNA and RNA) are capable of forming four-stranded structures named G-quadruplex- es (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 sup- portive 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 quadru- plex structures [22]. However, is not yet clear how the in vitro activities of this and other proteins on G-quad- ruplex DNA relate to their in vivo functions.
recombination analyses, we employed an in vitro system designed to detect intra- molecular homologous recombination events [8–11]. We the constructed two pBR322-based plasmids, recombinant substrates p73.1 and p80.1, bearing two copies cloned in the same orientation as the corre- sponding 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 the recombination that MsH43 inserts, 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 recombi- nation process.
In the present work, we also analyzed the effect of the presence of sequences capable of forming G-quad- ruplex structures on recombination frequency and the generation of recombinant molecules, taking advantage
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-
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P. Barros et al. Effect of cations and G-quartets on recombination
P. Barros et al. Effect of cations and G-quartets on recombination
Recombination substrates
P02.1
P02.2
P02.2
E P02.1
E
A ESc
E
S
MsH43 80.1
MsH43 73.1
E
E
MsH43 73.1
MsH43 80.1
p73.1
p80.1
ori
ori
MsH43 73.1
MsH43 80.1
E
E
E
B
Crossover
MsH43
LacZ
Pairing of homologous sequences
MsH43
Equal recombinant
Rec. substrate
X
Recombinant product (LacZ–)
ori
ori
MsH43
ori
Unequal recombinant
Amp
results of
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 con- served the original minisatellite sequence (data not shown). The
are
summarized
the experiments
(asterisks
variations
showed size
intramolecular homologous recombination in Table 1. The data were collected from three indepen- dent experiments for each assay condition (standard supplemented with salts). The LacZ+ colonies or were produced by the transformation with the origi- nal 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-inacti- vated extract (15 min, 100 (cid:2)C) was about 2 · 10)5. Under standard conditions, both the recombination frequencies and the frequency of equal and unequal recombinants were very similar with plasmids p73.1 the and p80.1. Noteworthy, with both plasmids, recombination events that generated unequal recombi- nants were more abundant ((cid:2) 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-
nant plasmids generated lacZ) bacteria (white colo- nies), and the original plasmids generated lacZ+ bacte- ria (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 con- taining the minisatellite) (Fig. 2A). If the recombinant was unequal, then the EcoRI fragment containing the in minisatellite Fig. 2A). In the case of the p80.1, the digestion with EcoRI of the original plasmid yielded four DNA frag- ments, whereas the digestion of its recombinant pro- (Fig. 2B). To ducts generated only one fragment 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 amplifi- cations 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 con- struct. Figure 2D shows the result of a heteroduplex assay employing the recombinants generated in the experiments with p80.1. The generation of hetero- duplex molecules denotes the presence of an unequal
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Fig. 1. Map of the recombination substrates and representation of an intramolecular homologous recombination event. (A) Plas- mids p73.1 and p80.1 (not drawn to scale) were used as substrates in the recombina- tion 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 homolo- gous recombination generates two kinds of plasmid: equals, in which the MsH43 remains unaltered, and unequals, with alter- ations in the minisatellite sequence.
P. Barros et al. Effect of cations and G-quartets on recombination
A
1
.
3 7 p
1
2
3
4
5 6
7
8
9
10
11
12 13 14 15
* * *
*
*
*
*
*
*
.
B
1 0 8 p
1
2
3
4 5 6
7
8
9
C
1 . 0 8 p
1
2
3
4
5 6
7
8
9
10
11
12 13 14
15
16
17 18 19 20
21 22 23
*
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*
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D
1 . 0 8 p
1
2
3
4
5 6
7
8
9
10
11
12 13 14 15
16
17
18
M
m o l e c u l e s
H e t e r o d u p l e x
the lize the G-quadruplex structure. Once again, recombination frequencies were very similar with both indicating that the capacity of the MsH43 plasmids,
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
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Fig. 2. Analysis of the recombinant prod- ucts generated in the in vitro recombination assays. (A) Analysis in 1.5% agarose gels of the EcoRI digest of the recombinant plas- mids obtained from lacZ) colonies in experi- ments 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 altera- tions in the size of MsH43 (unequal recomb- inants). (B) Analysis in 1.5% agarose gels of the EcoRI digest of the plasmids obtained from lacZ) colonies in recombination experi- ments 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 frag- ment 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 pres- ence 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).
P. Barros et al. Effect of cations and G-quartets on recombination
Table 1. 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, 6 and 8, mean ± stan- dard deviation of the data is provided.
Equal recombinants Unequal recombinants
Recombination substrate Assay conditions Recombination frequency (%) No. Frequency (%) No. Frequency (%) Colonies (LacZ+ ⁄ LacZ))
p73.1 Standard
16 21 19 26 24 31 3863 ⁄ 42 4034 ⁄ 45 4060 ⁄ 50
p80.1 Standard
35 28 29 45 40 45 7832 ⁄ 80 6095 ⁄ 68 6866 ⁄ 74
p73.1 20 mM KCl
24 28 23 15 19 12 8296 ⁄ 39 9183 ⁄ 47 7850 ⁄ 35
p80.1 20 mM KCl
25 35 31 14 12 13 9450 ⁄ 39 12 365 ⁄ 47 9617 ⁄ 44
p80.1 20 mM NaCl
23 21 16 6 6 5 19 559 ⁄ 29 17 360 ⁄ 27 16 261 ⁄ 21
p80.1 20 mM NH4Cl
recombination frequencies obtained with those cations were lower than with K+.
stability,
reduction (near 50%) in the recombination frequen- cies, as well as an important decrease in the nuclease activity of the nuclear extract (data not shown). As the initiation of homologous recombination is medi- ated by a nuclease activity that introduces DNA DSBs [23], it is possible that the reduction in recom- bination frequency was due to inhibition of the nucle- ase 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 condi- tions; that is, the equal recombinants were more abun- dant ((cid:2) 25%) than the unequal ones (Table 1). Was this inversion produced specifically by K+? The recom- bination assays carried out in the presence of Na+ or +, maintaining the same ionic strength, showed a NH4 marked reduction of the recombination frequency with respect to the standard conditions, more pronounced than with K+, and also a predominance of equal rec- + ombinants (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 the coherent explanation of
the observation that
Cations play essential roles in nucleic acid and pro- folding, and catalysis. By tein structure, 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 physio- logical 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 recom- bination 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 recombi- nants was higher. The presence of monovalent cations
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18 21 17 5 4 5 8114 ⁄ 23 8555 ⁄ 25 7962 ⁄ 22 1.087 1.116 1.232 1.145 ± 0.077 1.021 1.116 1.078 1.072 ± 0.048 0.470 0.512 0.448 0.476 ± 0.033 0.413 0.380 0.458 0.417 ± 0.039 0.148 0.156 0.129 0.144 ± 0.014 0.283 0.292 0.276 0.284 ± 0.008 0.414 0.521 0.468 0.468 ± 0.054 0.447 0.459 0.422 0.442 ± 0.018 0.289 0.305 0.293 0.296 ± 0.008 0.265 0.283 0.322 0.290 ± 0.029 0.118 0.121 0.098 0.112 ± 0.013 0.222 0.245 0.314 0.260 ± 0.048 0.673 0.595 0.764 0.677 ± 0.085 0.574 0.657 0.656 0.629 ± 0.048 0.181 0.207 0.155 0.181 ± 0.026 0.148 0.097 0.136 0.127 ± 0.027 0.030 0.035 0.031 0.032 ± 0.003 0.061 0.047 0.062 0.057 ± 0.008
reported for
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 con- formational changes in RAD51, leading to the forma- tion of interlinked recombination intermediates [2] that are essential for the correct progression of the recom- bination 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.
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 3 and 4) revealed that duplications occurred less frequently (30%) than deletions, similar to what was 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 conse- quence of simple direct duplications (Fig. 4), except in one case derived from p73.1, where one repeat was
P. Barros et al. Effect of cations and G-quartets on recombination
A
B
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Fig. 3. Organization of MsH43 recombinants containing deletions. (A) Sequence array of the deleted molecules obtained in experiments with + (N1–N4); asterisks indicate recombinants with p80.1: standard conditions (S1–S18), with K+ (K1–K21), with Na+ (Na1–Na9), and with NH4 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.
P. Barros et al. Effect of cations and G-quartets on recombination
B
A MsH43 80.1 allele
MsH43 73.1 allele
S1 S2
S3
S4
S5
S6
S7
S8
S9
S10
S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13
S11
S12
S13
S14
S15
K1
K2
K3
K4
K5
K6
K7
K8
K1 K2 K3 K4 K5 K6 K7 K8 K9 K10 K11 K12 K13 K14
K9
K10
K11
K12
K13
K14
K15
Na1 Na2 Na3 Na4 Na5 Na6 Na7
K16
K17
K18
K19
N1 N2 N3 N4 N5 N6 N7
leading to duplications in
the recombination events, the minisatellite sequence.
that
There is an emerging consensus
Interestingly,
the G-quadruplex motif
secondary structures of DNA have the potential to induce geno- mic instability. The role of nonlinear DNA in replica- recombination and transcription has become tion, 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 struc- tural motifs as signals for processes such as gene regu- lation 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
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 reorganiza- tions produced in the minisatellite MsH43 arose by a homology-guided mechanism. in the presence of K+, the p80.1 recombinants displaying duplications did not include the repeats located at the 5¢-side of (K1–K14 in Fig. 4A). In contrast, this limitation was not found in the assays carried out either under standard conditions +, or with p73.1 or in the presence of Na+ or NH4 (Fig. 4). It is tempting to speculate that the G-quadru- plex motif (TGGGGC)4 influences the resolution of
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Fig. 4. 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.
P. Barros et al. Effect of cations and G-quartets on recombination
(TGGGGC)4
A
´ 5´
5´ ´
DSB
B
5´
C
3´ 5´
5´ 3´
G4 structure
5´
5´
Slippage loop
Slippage loop
3´
3´
5´
5´
activities [30]. Furthermore, G-quadruplex secondary structures can induce genetic rearrangements and pro- mote RecA-independent homologous recombination [31]. Genome-wide predictions have shown an abun- dance 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 progres- sion of DNA polymerases [34,35].
this
type of
the
inclusion of
the
this was tion assay formed G-quadruplex, or that unstable. In both cases, the slippage process would not be interfered with by the G-quadruplex structure, making possible sequence (TGGGGC)4 in some recombinant molecules. Support- ing 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 con- centrations of 100 mm K+, there is a residual amount of oligonucleotides that do not form G-quadruplex.
Possible
The present work shows that the presence of mono- valent 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 that point. leading to blockage of polymerases at Therefore, the G-quadruplex would not be a stimulus for recombination but a source of genomic instability.
Our results demonstrate that a minisatellite sequence, which is not included inside a gene [12], can form G- quadruplex structures that interfere with DNA synthesis and influence the resolution of recombination. It is tempting to speculate that repetitive sequence could be involved in processes related to gen- ome stability. In this regard, although the mechanisms involved in minisatellite instability are poorly under- stood, some relevant factors have already been found, such as the requirement for DSBs [23] and length and sequence heterozygosity [36]. Furthermore, size altera- tions 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-quad- ruplex motif of MsH43, suggesting that this structure is involved in the recombination events leading to duplica- tions. 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. Accord- ing 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 replica- tion at the 5¢-end of the G-quadruplex motif and conse- quently 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-quad- ruplex 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 in the duplicated contains the G-quadruplex motif for explanations (Fig. 4A). sequence the generation of the recombinant K1 could be that not all p80.1 plasmid molecules present in the recombina-
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Fig. 5. Recombination model involving G-quadruplex (G4) structure and slippage processes for the generation of recombinant mole- cules containing duplications and deletions. (A) Generation of a DSB by the nuclease(s) of the nuclear extract initiates the recombi- nation process; the sequence (TGGGGC)4 represents the G-quadru- plex 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 con- taining the G-quadruplex motif, a G-quadruplex structure could be generated that would be stabilized by the presence of K+; this sec- ondary 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 gen- erated without the interference of G-quadruplex structures. Accord- it is worth noting that the slippage in the ing to this model, 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.
the results obtained with It should be noted that 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 neces- sary 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 recombi- nation and DNA instability, as well for the analysis of how monovalent cations affect the proteins implicated in this fundamental biological process.
nuclear extracts for 30 min at 37 (cid:2)C, DNA was phenol- extracted, 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 gÆL)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 fre- quency 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 (cid:2)C) was about 2 · 10)5. The white colonies (recombinant products) were used for minipreparation of plasmid DNA and aliquots of (cid:2) 300 ng were digested with EcoRI, and analyzed in agarose gels.
Experimental procedures
PCR, DNA sequencing, and heteroduplex analysis
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, Madi- son, 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
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 (cid:2)C for 1 min, 56 (cid:2)C for 30 s, and 72 (cid:2)C for 40 s, and a final cycle with an extension of 5 min. 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 prod- ucts were cycle sequenced by 25 cycles of 96 (cid:2)C for 10 s and 68 (cid:2)C for 2 min 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 5 lL of the PCR products obtained from the recombinants and from the ori- ginal recombination substrates were mixed at 95 (cid:2)C for 3 min, and slowly cooled to room temperature. The hetero- duplex molecules were detected by electrophoresis in 5% polyacrylamide gels (29 : 1) at a constant voltage of 140 V for 6 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 Educacio´ n y Ciencia (BFU2006-06708) and by the Xunta de Galicia (PGIDT07PX12001099R).
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