Potential G-quadruplex formation at breakpoint regions of chromosomal translocations in cancer may explain their fragility

Genetic alterations like point mutations, insertions, deletions, inversions and translocations are frequently found in cancers. Chromosomal translocations are one of the most common genomic aberrations associated with nearly all types of cancers especially leukemia and lymphoma. Recent studies have shown the role of non-B DNA structures in generation of translocations. In the present study, using various bioinformatic tools, we show the propensity of formation of different types of altered DNA structures near translocation breakpoint regions. In particular, we find close association between occurrence of G-quadruplex forming motifs and fragile regions in almost 70% of genes involved in rearrangements in lymphoid cancers. However, such an analysis did not provide any evidence for the occurrence of G-quadruplexes at the close vicinity of translocation breakpoint regions in nonlymphoid cancers. Overall, this study will help in the identification of novel non-B DNA targets that may be responsible for generation of chromosomal translocations in cancer.     

S1 nuclease hypersensitive sites in an oligopurine/oligopyrimidine DNA from the t(10;14) breakpoint cluster region.

Recurring chromosomal translocations are frequently seen in cancers, especially in leukemias and lymphomas. The genes affected by these chromosomal translocations appear to play an important role in oncogenesis. The mechanism underlying the formation of chromosomal translocation is a subject under extensive study. In chromosomal translocations involving the Ig and TCR loci, complete heptamer-spacer-nonamer signal motifs are usually present at the break of the Ig and TCR genes, indicating the involvement of V-D-J recombinase(s). On the other hand, in only about 50% of the cases signal motif sequences have been found at the break in the other participating chromosome, suggesting that different mechanisms may be involved in the scission of the corresponding chromosome. Here we report the identification of an oligopurine/oligopyrimidine DNA in the t(10;14) breakpoint cluster region associated with T-cell acute lymphoblastic leukemia. S1 nuclease mapping revealed multiple S1 hypersensitive sites in the oligopurine/oligopyrimidine DNA. These data suggest a role for oligopurine/oligopyrimidine sequences (non-B DNA) in the formation of chromosomal translocation.     

The structure-specific nicking of small heteroduplexes by the RAG complex: implications for lymphoid chromosomal translocations

During V(D)J recombination, the RAG complex binds at recombination signal sequences and creates double-strand breaks. In addition to this sequence-specific recognition of the RSS, the RAG complex has been shown to be a structure-specific nuclease, cleaving 3' overhangs and 3' flaps, and, more recently, 10 nucleotides (nt) bubble (heteroduplex) structures. Here, we assess the smallest size heteroduplex that core and full-length RAGs can cleave. We also test whether bubbles adjacent to a partial RSS are nicked any differently or any more efficiently than bubbles that are surrounded by random sequence. These points are important in considering what types and what size of non-B DNA structure that the RAG complex can nick, and this helps assess the role of the RAG complex in mediating lymphoid chromosomal translocations. We find that the smallest bubble nicked by the RAG complex is 3nt, and proximity to a partial or full RSS sequence does not affect the nicking by RAGs. RAG nicking efficiency increases with the size of the heteroduplex and is only about two-fold less efficient than an RSS when the bubble is 6nt. We consider these findings in the context of RAG nicking at non-B DNA structures in lymphoid chromosomal translocations.     

Non-B DNA conformations, mutagenesis and disease

Recent discoveries have revealed that simple repeating DNA sequences, which are known to adopt non-B DNA conformations (such as triplexes, cruciforms, slipped structures, left-handed Z-DNA and tetraplexes), are mutagenic. The mutagenesis is due to the non-B DNA conformation rather than to the DNA sequence per se in the orthodox right-handed Watson-Crick B-form. The human genetic consequences of these non-B structures are approximately 20 neurological diseases, approximately 50 genomic disorders (caused by gross deletions, inversions, duplications and translocations), and several psychiatric diseases involving polymorphisms in simple repeating sequences. Thus, the convergence of biochemical, genetic and genomic studies has demonstrated a new paradigm implicating the non-B DNA conformations as the mutagenesis specificity determinants, not the sequences as such.     

Formation of a G-quadruplex at the BCL2 major breakpoint region of the t(14;18) translocation in follicular lymphoma

The t(14;18) translocation in follicular lymphoma is one of the most common chromosomal translocations. Most breaks on chromosome 18 are located at the 3'-UTR of the BCL2 gene and are mainly clustered in the major breakpoint region (MBR). Recently, we found that the BCL2 MBR has a non-B DNA character in genomic DNA. Here, we show that single-stranded DNA modeled from the template strand of the BCL2 MBR, forms secondary structures that migrate faster on native PAGE in the presence of potassium, due to the formation of intramolecular G-quadruplexes. Circular dichroism shows evidence for a parallel orientation for G-quadruplex structures in the template strand of the BCL2 MBR. Mutagenesis and the DMS modification assay confirm the presence of three guanine tetrads in the structure. 1H nuclear magnetic resonance studies further confirm the formation of an intramolecular G-quadruplex and a representative model has been built based on all of the experimental evidence. We also provide data consistent with the possible formation of a G-quadruplex structure at the BCL2 MBR within mammalian cells. In summary, these important features could contribute to the single-stranded character at the BCL2 MBR, thereby contributing to chromosomal fragility.     

Chromosomal translocations in cancer

Genetic alterations in DNA can lead to cancer when it is present in proto-oncogenes, tumor suppressor genes, DNA repair genes etc. Examples of such alterations include deletions, inversions and chromosomal translocations. Among these rearrangements chromosomal translocations are considered as the primary cause for many cancers including lymphoma, leukemia and some solid tumors. Chromosomal translocations in certain cases can result either in the fusion of genes or in bringing genes close to enhancer or promoter elements, hence leading to their altered expression. Moreover, chromosomal translocations are used as diagnostic markers for cancer and its therapeutics. In the first part of this review, we summarize the well-studied chromosomal translocations in cancer. Although the mechanism of formation of most of these translocations is still unclear, in the second part we discuss the recent advances in this area of research.     

Yeast Pol4 Promotes Tel1-Regulated Chromosomal Translocations

DNA double-strand breaks (DSBs) are one of the most dangerous DNA lesions, since their erroneous repair by nonhomologous end-joining (NHEJ) can generate harmful chromosomal rearrangements. PolX DNA polymerases are well suited to extend DSB ends that cannot be directly ligated due to their particular ability to bind to and insert nucleotides at the imperfect template-primer structures formed during NHEJ. Herein, we have devised genetic assays in yeast to induce simultaneous DSBs in different chromosomes in vivo. The repair of these breaks in trans could result in reciprocal chromosomal translocations that were dependent on classical Ku-dependent NHEJ. End-joining events leading to translocations were mainly based on the formation of short base pairing between 3′-overhanging DNA ends coupled to gap-filling DNA synthesis. A major proportion of these events were specifically dependent on yeast DNA polymerase Pol4 activity. In addition, we have discovered that Pol4-Thr⁵⁴⁰ amino acid residue can be phosphorylated by Tel1/ATM kinase, which could modulate Pol4 activity during NHEJ. Our data suggest that the role of Tel1 in preventing break-induced chromosomal translocations can, to some extent, be due to its stimulating effect on gap-filling activity of Pol4 to repair DSBs in cis. Overall, this work provides further insight to the molecular mechanisms of DSB repair by NHEJ and presents a new perspective to the understanding of how chromosomal translocations are formed in eukaryotic cells.     

The involvement of non-B DNA structures in gross chromosomal rearrangements

Non-B DNA conformations adopted by certain types of DNA sequences promote genetic instabilities, especially gross rearrangements including translocations. We conclude the following: (a) slipped (hairpin) structures, cruciforms, triplexes, tetraplexes and i-motifs, and left-handed Z-DNA are formed in chromosomes and elicit profound genetic consequences via recombination-repair, (b) repeating sequences, probably in their non-B conformations, cause gross genomic rearrangements (translocations, deletions, insertions, inversions, and duplications), and (c) these rearrangements are the genetic basis for numerous human diseases including polycystic kidney disease, adrenoleukodystrophy, follicular lymphomas, and spermatogenic failure.     

118 DNA structure-induced genetic instability in mammals

Naturally occurring repetitive DNA sequences can adopt alternative (i.e. non-B) DNA secondary structures, and often co-localize with chromosomal breakpoint “hotspots,” implicating non-B DNA in translocation-related cancer etiology. We have found that sequences capable of adopting H-DNA and Z-DNA structures are intrinsically mutagenic in mammals. For example, an endogenous H-DNA-forming sequence from the human c-MYC promoter and a model Z-DNA-forming CpG repeat induced genetic instability in mammalian cells, largely in the form of deletions resulting from DNA double-strand breaks (Wang & Vasquez, 2004; Wang et al., 2006). Characterization of the mutants revealed microhomologies at the breakpoints, consistent with a microhomology-mediated end-joining repair of the double-strand breaks (Kha et al., 2010). We have constructed transgenic mutation-reporter mice containing these human H-DNA- and Z-DNA-forming sequences to determine their effects on genomic instability in a chromosomal context in a living organism (Wang et al., 2008). Initial results suggest that both H-DNA- and Z-DNA-forming sequences induced genetic instability in mice, suggesting that these non-B DNA structures represent endogenous sources of genetic instability and may contribute to disease etiology and evolution. Our current studies are designed to determine the mechanisms of DNA structure-induced genetic instability in mammals; the roles of helicases, polymerases, and repair enzymes in H-DNA and Z-DNA-induced genetic instability will be discussed.     

The Human Specialized DNA Polymerases and Non-B DNA: Vital Relationships to Preserve Genome Integrity

In addition to the canonical right-handed double helix, DNA molecule can adopt several other non-B DNA structures. Readily formed in the genome at specific DNA repetitive sequences, these secondary conformations present a distinctive challenge for progression of DNA replication forks. Impeding normal DNA synthesis, cruciforms, hairpins, H DNA, Z DNA and G4 DNA considerably impact the genome stability and in some instances play a causal role in disease development. Along with previously discovered dedicated DNA helicases, the specialized DNA polymerases emerge as major actors performing DNA synthesis through these distorted impediments. In their new role, they are facilitating DNA synthesis on replication stalling sites formed by non-B DNA structures and thereby helping the completion of DNA replication, a process otherwise crucial for preserving genome integrity and concluding normal cell division. This review summarizes the evidence gathered describing the function of specialized DNA polymerases in replicating DNA through non-B DNA structures.     

Chromosomal translocations involving the MLL gene: molecular mechanisms

A wide array of recurrent, non-random chromosomal translocations are associated with hematologic malignancies; experimental models have clearly demonstrated that many of these translocations are causal events during malignant transformation. Translocations involving the MLL gene are among the most common of these non-random translocations. Leukemias with MLL translocations have been the topic of intense interest because of the unusual, biphenotypic immunophenotype of these leukemias, because of the unique clinical presentation of some MLL translocations (infant leukemia and therapy-related leukemia), and because of the large number of different chromosomal loci that partner with MLL in these translocations. This review is focused on the potential mechanisms that lead to MLL translocations, and will discuss aberrant VDJ recombination, Alu-mediated recombination, non-homologous end joining, as well as the effect of DNA topoisomerase II poisons and chromatin structure.     

Site-specific chromosomal rearrangements induced in human diploid cells by x-irradiation

A total of 130 stable, two-break reciprocal translocations were scored in G-banded karyotypes prepared from 375 metaphase spreads from a strain of human diploid fibroblasts irradiated with 400 or 600 rads and analyzed 1-20 mean population doublings later. The chromosomal location of each of the 260 breakpoints was mapped. The sites of 121 chromosomal breaks and deletions in the first postirradiation mitosis were also scored. Unlike the random distribution of these latter events, the translocation breakpoints showed not only a nonrandom distribution among chromosomes but also the existence of specific sites within chromosomes that were more frequently involved in translocations. The most notable finding was a marked excess of translocations involving the short arm of chromosome 1, in particular, band 1p22. The specific types of translocations were random, although the breakpoints were not. Eight of the 12 most frequently involved chromosomal sites were regions in which fragile sites have been mapped in human lymphocytes.     

Non-B DNA structure-induced genetic instability

Repetitive DNA sequences are abundant in eukaryotic genomes, and many of these sequences have the potential to adopt non-B DNA conformations. Genes harboring non-B DNA structure-forming sequences increase the risk of genetic instability and thus are associated with human diseases. In this review, we discuss putative mechanisms responsible for genetic instability events occurring at these non-B DNA structures, with a focus on hairpins, left-handed Z-DNA, and intramolecular triplexes or H-DNA. Slippage and misalignment are the most common events leading to DNA structure-induced mutagenesis. However, a number of other mechanisms of genetic instability have been proposed based on the finding that these structures not only induce expansions and deletions, but can also induce DNA strand breaks and rearrangements. The available data implicate a variety of proteins, such as mismatch repair proteins, nucleotide excision repair proteins, topoisomerases, and structure specific-nucleases in the processing of these mutagenic DNA structures. The potential mechanisms of genetic instability induced by these structures and their contribution to human diseases are discussed.     

Insertion of L1 elements into sites that can form non-B DNA. Interactions of non-B DNA-forming sequences

Three rat L1 element integration (target) sites chosen at random can adopt non-B DNA structures in vitro at normal bacterial superhelical densities. These target sites contain, respectively, short, mixed (AT)n tracts that we show can form one or more cruciforms, short (GT)n tracts, or polypurine:polypyrimidine regions. These sites share no sequence homology, and a non-B DNA structure appears to be the only feature common to them all. When the right end of the L1Rn3 element which forms a complex series of non-B DNA structures including two triplexes, and its target site which undergoes cruciform extrusion, are present on the same supercoiled molecule, they compete for available supercoil energy. The amount of non-B DNA formed at each site varies with pH, the concentration of cations, and the size of the topological domain. The implication of our findings for recombination of L1 elements and for the effect of these elements on contiguous DNA sequences is discussed.     

Double-strand break formation by the RAG complex at the bcl-2 major breakpoint region and at other non-B DNA structures in vitro

The most common chromosomal translocation in cancer, t(14;18) at the 150-bp bcl-2 major breakpoint region (Mbr), occurs in follicular lymphomas. The bcl-2 Mbr assumes a non-B DNA conformation, thus explaining its distinctive fragility. This non-B DNA structure is a target of the RAG complex in vivo, but not because of its primary sequence. Here we report that the RAG complex generates at least two independent nicks that lead to double-strand breaks in vitro, and this requires the non-B DNA structure at the bcl-2 Mbr. A 3-bp mutation is capable of abolishing the non-B structure formation and the double-strand breaks. The observations on the bcl-2 Mbr reflect more general properties of the RAG complex, which can bind and nick at duplex-single-strand transitions of other non-B DNA structures, resulting in double-strand breaks in vitro. Hence, the present study reveals novel insight into a third mechanism of action of RAGs on DNA, besides the standard heptamer/nonamer-mediated cleavage in V(D)J recombination and the in vitro transposase activity.     

Detection of G-Quadruplex DNA Using Primer Extension as a Tool

DNA sequence and structure play a key role in imparting fragility to different regions of the genome. Recent studies have shown that non-B DNA structures play a key role in causing genomic instability, apart from their physiological roles at telomeres and promoters. Structures such as G-quadruplexes, cruciforms, and triplexes have been implicated in making DNA susceptible to breakage, resulting in genomic rearrangements. Hence, techniques that aid in the easy identification of such non-B DNA motifs will prove to be very useful in determining factors responsible for genomic instability. In this study, we provide evidence for the use of primer extension as a sensitive and specific tool to detect such altered DNA structures. We have used the G-quadruplex motif, recently characterized at the BCL2 major breakpoint region as a proof of principle to demonstrate the advantages of the technique. Our results show that pause sites corresponding to the non-B DNA are specific, since they are absent when the G-quadruplex motif is mutated and their positions change in tandem with that of the primers. The efficiency of primer extension pause sites varied according to the concentration of monovalant cations tested, which support G-quadruplex formation. Overall, our results demonstrate that primer extension is a strong in vitro tool to detect non-B DNA structures such as G-quadruplex on a plasmid DNA, which can be further adapted to identify non-B DNA structures, even at the genomic level.