Francisco Boán

Inhibition of DNA synthesis by K + -stabilised G-quadruplex promotes allelic preferential amplification

PCR preferential amplification consists of the inefficient amplification of one allele in a heterozygous sample. Here, we report the isolation of a GC‐rich human minisatellite, MsH43, that undergoes allelic preferential amplification during PCR. This effect requires the existence of a (TGGGGC)4 motif that is able to form a G‐quadruplex in the presence of K+. This structure interferes with the DNA synthesis of the alleles harbouring this motif during PCR The present results are the first demonstration that the formation of G‐quadruplex can be one of the mechanisms involved in some kinds of preferential amplification.

MOLECULAR CHARACTERIZATION OF A NEW HUMAN MINISATELLITE THAT IS ABLE TO FORM SINGLE-STRANDED LOOPS IN VITRO AND IS RECOGNIZED BY NUCLEAR PROTEINS

We report the isolation of a new low polymorphic GC‐rich human minisatellite locus (MsH42) that contains different recombination motifs and is homologous to sequences involved in immunoglobulin class‐switching. Furthermore, we show that MsH42 undergoes slipped‐strand mispairing during PCR indicating its ability to generate single‐stranded loops. Specific DNA‐protein complexes were detected in band‐shifting experiments using nuclear extracts from mouse testes and human NC‐37 cells. The possible implications of this minisatellite in recombination events is discussed.

Effect of monovalent cations and G-quadruplex structures on the outcome of intramolecular homologous recombination.

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.

A paradox in the in vitro end-joining assays.

Much work has been focused on the pathways that restore the integrity of the genome after different kinds of lesions, especially double-strand breaks. A classical method to investigate double-strand break repair is the incubation of a DNA substrate with cell-free extracts. In these end-joining assays, the DNA is efficiently ligated by the proteins present in the extract, generating circular molecules and/or multimers. In contrast, using a similar in vitro system, we detected DNA cleavage rather than end ligation. When comparing our results with previous works, a paradox emerges: lower amounts of DNA become multimerized instead of degraded and higher amounts of DNA are degraded rather than multimerized. Here, we have demonstrated that when the DNA/protein ratio is low enough, the DNA-binding proteins of the nuclear extract protect the DNA substrate, avoiding DNA degradation and vice versa. Therefore, the variation of the DNA/protein ratio is enough to switch the outcome of the experiment from a DNA cleavage assay to a typical end-joining assay.

In Vitro Recombination Mediated by G-Quadruplexes

Repetitive DNA is very abundant in genomes and can adopt several types of non-B DNA secondary structures. 2] Among them are the guanine-rich repeats, which have the capacity to form G-quadruplexes (G4). G4 DNA is a structure in which intraor interstrand interactions are stabilized by G quartets, which are planar arrays of four guanines paired by Hoogsteen bonding. 4] In the human genome, the number of sites with potential for formation of G4 structures might look high (over 370 000) but this figure is lower than expected, except in promoters and introns. Moreover, recent advances have established how this DNA structure might contribute to processes such as gene regulation, genomic instability and recombination in eukaryotic cells. Here we show that the interaction of G4 motifs provokes the formation of recombinant molecules in vitro using two copies of the G-rich human minisatellite MsH43 as the recombination target. This process only occurs when both MsH43 copies are cloned at inverted orientation. To analyze how the presence of G4 motifs could influence the formation of recombinant molecules during PCR, we used the G-rich human minisatellite MsH43, which is composed of eleven different repeat units of 5–6 bp. Most of its alleles contain the sequence TGGGGC repeated four times in tandem (Figure 1, green repeats) ; this motif can form a G4 structure in the presence of K and interferes with DNA synthesis during PCR. We took advantage of the finding that the MsH43 80.1 allele has the capability to adopt a G4 structure whereas the 73.1 allele does not to monitor the possible influence of this secondary structure in the generation of recombinant molecules during PCR. We employed plasmids carrying two copies of a DNA fragment containing either the MsH43 80.1 allele or the MsH43 73.1. In each plasmid, both DNA fragments were cloned either in the same (p80.1d and p73.1d) or inverted orientation (p80.1i and p73.1i). Figure 1 shows a map of the plasmids, p73.1 and p80.1, as well as the repeat organization of the 80.1 and 73.1 MsH43 alleles. Plasmids were subjected to PCR analysis with primers 5e/3e, which flank the copy of MsH43 upstream of the lacZ gene, and 5a/3a, which flank the second copy of MsH43 located downstream of the lacZ gene (Figure 1). Despite the propensity of minisatellites to produce PCR artifacts due to the presence of the same or similar motifs in templates, the analysis of PCR products showed that they corresponded to the expected bands and no artifacts were observed (Figure 2). The DNA fragment generated with 5e/3e ( 750 bp) is slightly larger than the product obtained with 5a/ 3a ( 720 bp) because 5e/3e includes a bigger portion of the minisatellite flanking sequence. In the experiments carried out with primers 5e/5a, which should not render amplification products because they are in the same strand, we did not obtain amplification with p80.1d, p73.1d and p73.1i. In contrast, p80.1i, which harbors two copies of the allele 80.1 at inverted orientation, generated an amplification product of ~450 bp (Figure 2). Likewise, the amplification of these plasmids with primers 3e/3a only generated a product of ~435 bp in the case of p80.1i. This in vitro recombination does not depend on the circularity of the plasmid because p80.1i linearized with AlwNI also produced the recombinant product (Figure 2). The sequencing of the amplification products obtained with 5e/3e corroborated that the minisatellite was not altered during PCR reactions. Sequence analysis of the PCR recombinant products obtained with 5e/5a revealed that this DNA fragment presents a deletion of 52 repeats, which correspond to repeats 23–74 in the original MsH43 80.1 allele (Figure 3). In this recombinant minisatellite, the 5’ flanking sequence and the initial 22 repeats correspond to the copy upstream of lacZ, whereas the last six repeats and the 3’ flanking sequence belongs to the copy downstream of lacZ. The analysis of the PCR product obtained with primers 3e/3a indicated that it was also generated by a recombination event that included the same portions of the two minisatellite copies (data not shown). In conclusion, the generation of the recombinant product depends on two factors: 1) the orientation of the two minisatellite copies in the plasmid since p80.1d does not generate any amplification product, and 2) the presence of the G4 motif since the p73.1 (d or i) did not form the recombinant product. Analyses of the sequences of both MsH43 alleles by using the Quadparser algorithm and the Quadfinder server confirmed that the 73.1 allele is entirely devoid of G4 structures and that the sole sequence in the 80.1 allele with the capacity of forming G-quadruplex structures is the motif (TGGGGC)4 (data not shown). The PCR recombination process can be affected by different factors, such as the formation of incomplete PCR products, the number of cycles, the existence of nicked DNA, the type of DNA polymerase or the bacterial host. 15] However, none of these factors seem to be responsible for the generation of the recombinant product obtained in the PCR reactions (data not shown). It could be that the recombinant molecule was the result of a “jumping reaction” favored by the presence of two copies of a repetitive sequence as it was reported for Alu sequences. Once again this situation seems not to be the case because the generation of the recombinant molecule was only observed with plasmid p80.1i ; this indicates that both the in[a] Dr. F. Bo n, Prof. Dr. J. G mez-M rquez Departamento de Bioqu mica e Biolox a Molecular Facultade de Biolox a-CIBUS Universidade de Santiago de Compostela 15782 Santiago, Galicia (Spain) Fax: (+ 34) 981528006 E-mail : jaime.gomez.marquez@usc.es

A new EcoRI family of satellite DNA in lampreys

Satellite DNA sequences have been studied in several groups of organisms. However, until now this type of sequence has not been characterized in cyclostomata, an evolutionarily important class of vertebrates. In the present work, we report the molecular characterization of a new family of satellite DNA in lampreys (Petromyzon marinus). Digestion of lamprey DNA with EcoRI identified a series of very abundant AT‐rich (60% A+T) repeating units, with short stretches of AT, that are multimers of 370 bp. Southern blot analysis and comparison with the satellite DNA sequences deposited in the databases indicate that this new family of satellite DNA is exclusive to lampreys. The distribution of this EcoRI satellite DNA on lamprey chromosomes was analyzed by in situ hybridization. The evolutionary origin of this satellite is briefly discussed.

DNA end-joining driven by microhomologies catalyzed by nuclear extracts

Abstract In a previous work we used an in vitro system for the generation and analysis of double-strand breaks (DSBs) using nuclear extracts from rat testes as a source of DSB activity. Since the recombination process can be triggered by the formation of DSB, in the present study we developed a strategy to isolate and characterize recombinant molecules using the same in vitro system. Our results indicate that the mechanism for the formation of recombinants was non-homologous end-joining driven by microhomologies. The procedure described here represents an alternative to investigate the mechanisms of DNA end-joining and other forms of DNA repair.