The Drosophila chk2 gene loki is essential for embryonic DNA double-strand-break checkpoints induced in S phase or G2

Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the occurrence of the next cell cycle transition. The Chk2 family of kinases is known to play a central role in mediating the cellular responses to DNA damage or DNA replication blocks in various organisms. Here we show through a phylogenetic study that the Drosophila melanogaster serine/threonine kinase Loki is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses allowed us to conclude that, in flies, chk2 is involved in monitoring double-strand breaks (DSBs) caused by irradiation during S and G2 phases. In this process it plays an essential role in inducing a cell cycle arrest in embryonic cells. Our results also show that, in contrast to C. elegans chk2, Drosophila chk2 is not essential for normal meiosis and recombination, and it also appears to be dispensable for the MMS-induced DNA damage checkpoint and the HU-induced DNA replication checkpoint during larval development. In addition, Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. As mutations in human chk2 were linked to several cancers, these similarities point to the usefulness of the Drosophila model system.

REV7 counteracts DNA double-strand break resection and affects PARP inhibition

Error-free repair of DNA double-strand breaks (DSBs) is achieved by homologous recombination (HR), and BRCA1 is an important factor for this repair pathway. In the absence of BRCA1-mediated HR, the administration of PARP inhibitors induces synthetic lethality of tumour cells of patients with breast or ovarian cancers. Despite the benefit of this tailored therapy, drug resistance can occur by HR restoration. Genetic reversion of BRCA1-inactivating mutations can be the underlying mechanism of drug resistance, but this does not explain resistance in all cases. In particular, little is known about BRCA1-independent restoration of HR. Here we show that loss of REV7 (also known as MAD2L2) in mouse and human cell lines re-establishes CTIP-dependent end resection of DSBs in BRCA1-deficient cells, leading to HR restoration and PARP inhibitor resistance, which is reversed by ATM kinase inhibition. REV7 is recruited to DSBs in a manner dependent on the H2AX-MDC1-RNF8-RNF168-53BP1 chromatin pathway, and seems to block HR and promote end joining in addition to its regulatory role in DNA damage tolerance. Finally, we establish that REV7 blocks DSB resection to promote non-homologous end-joining during immunoglobulin class switch recombination. Our results reveal an unexpected crucial function of REV7 downstream of 53BP1 in coordinating pathological DSB repair pathway choices in BRCA1-deficient cells.

Intravenous Iodinated Contrast Agents Amplify DNA Radiation Damage at CT

PURPOSE To determine the effect of the use of iodinated contrast agents on the formation of DNA double-strand breaks during chest computed tomography (CT). MATERIALS AND METHODS This study was approved by the institutional review board, and written informed consent was obtained from all patients. This single-center study was performed at a university hospital. A total of 179 patients underwent contrast material-enhanced CT, and 66 patients underwent unenhanced CT. Blood samples were taken from these patients prior to and immediately after CT. In these blood samples, the average number of phosphorylated histone H2AX (γH2AX) foci per lymphocyte was determined with fluorescence microscopy. Significant differences between the number of foci that developed in both the presence and the absence of the contrast agent were tested by using an independent sample t test. RESULTS γH2AX foci levels were increased in both groups after CT. Patients who underwent contrast-enhanced CT had an increased amount of DNA radiation damage (mean increase ± standard error of the mean, 0.056 foci per cell ± 0.009). This increase was 107% ± 19 higher than that in patients who underwent unenhanced CT (mean increase, 0.027 foci per cell ± 0.014). CONCLUSION The application of iodinated contrast agents during diagnostic x-ray procedures, such as chest CT, leads to a clear increase in the level of radiation-induced DNA damage as assessed with γH2AX foci formation.

DNA-DAMAGING REACTIONS OF NEOCARZINOSTATIN (ANTIBIOTICS, DNA REPAIR, CYTOTOXICITY)

Sensitive assays utilizing a cell-free and an intracellular system were employed to study the molecular bases of the DNA-damaging reactions of neocarzinostatin (NCS). In the cell-free DNA system, super-helical form I DNA from the bacteriophage PM2 was used as the substrate. The three forms of DNA present after treatment with NCS were separated by agarose gel electrophoresis. When NCS-damaged DNA was assayed under neutral conditions, there was a progressive decrease in the amount of surviving form I DNA and a corresponding increase in form II (nicked, relaxed circular) DNA, but very little increase in form III (linear duplex) DNA. This indicates that NCS introduces primarily single-strand breaks. However later studies showed that there were some site-specific double-strand breaks mediated by NCS on PM2 DNA. Seven such specific sites were mapped on the PM2 genome. When the damage was assayed under nondenaturing alkaline conditions or with the apurinic/apyrimidinic endonuclease IV, there was a slightly greater decrease in the amount of surviving form I DNA compared with neutral conditions indicating the presence of some alkali-labile sites.^ NCS-mediated DNA damage and repair were examined with cultured Chinese hamster ovary (CHO) cells using either alkaline elution for analysis of single-strand breaks or neutral elution for analysis of double-strand breaks. Most of the strand breaks introduced by NCS were capable of being rejoined. However, there was a small amount of residual DNA damage remaining unrejoined at 24-hr after removal of the drug. The amount of residual DNA damage was higher in a CHO mutant cell line (EM9) having a higher sensitivity to killing by NCS than its parental strain (AA8). Other lesions, DNA-protein complexes and alkali-labile sites, were detected after NCS treatment but they constituted only a small fraction of the DNA damage.^ Based on the above information, it can be postulated that NCS introduces some very lethal DNA damage. It is likely that the lethal lesions are a subset of the total DNA lesions representing the residual DNA damage. This DNA damage may be composed of site-specific, unrejoinable double-strand breaks and are thus the primary lesion leading to NCS-mediated lethality.^

STUDIES OF THE MOLECULAR MECHANISMS OF CHROMOSOME ABERRATION FORMATION (DNA REPAIR, CROSSLINKS, MITOMYCIN C)

The objective of this research has been to study the molecular basis for chromosome aberration formation. Predicated on a variety of data, Mitomycin C (MMC)-induced DNA damage has been postulated to cause the formation of chromatid breaks (and gaps) by preventing the replication of regions of the genome prior to mitosis. The basic protocol for these experiments involved treating synchronized Hela cells in G(,1)-phase with a 1 (mu)g/ml dose of MMC for one hour. After removing the drug, cells were then allowed to progress to mitosis and were harvested for analysis by selective detachment. Utilizing the alkaline elution assay for DNA damage, evidence was obtained to support the conclusion that Hela cells can progress through S-phase into mitosis with intact DNA-DNA interstrand crosslinks. A higher level of crosslinking was observed in those cells remaining in interphase compared to those able to reach mitosis at the time of analysis. Dual radioisotope labeling experiments revealed that, at this dose, these crosslinks were associated to the same extent with both parental and newly replicated DNA. This finding was shown not to be the result of a two-step crosslink formation mechanism in which crosslink levels increase with time after drug treatment. It was also shown not to be an artefact of the double-labeling protocol. Using neutral CsCl density gradient ultracentrifugation of mitotic cells containing BrdU-labeled newly replicated DNA, control cells exhibited one major peak at a heavy/light density. However, MMC-treated cells had this same major peak at the heavy/light density, in addition to another minor peak at a density characteristic for light/light DNA. This was interpreted as indicating either: (1) that some parental DNA had not been replicated in the MMC treated sample or; (2) that a recombination repair mechanism was operational. To distinguish between these two possibilities, flow cytometric DNA fluorescence (i.e., DNA content) measurements of MMC-treated and control cells were made. These studies revealed that the mitotic cells that had been treated with MMC while in G(,1)-phase displayed a 10-20% lower DNA content than untreated control cells when measured under conditions that neutralize chromosome condensation effects (i.e., hypotonic treatment). These measurements were made under conditions in which the binding of the drug, MMC, was shown not to interfere with the stoichiometry of the ethidium bromide-mithramycin stain. At the chromosome level, differential staining techniques were used in an attempt to visualize unreplicated regions of the genome, but staining indicative of large unreplicated regions was not observed. These results are best explained by a recombinogenic mechanism. A model consistent with these results has been proposed.^

LOCALIZATION OF DNA REPAIR FUNCTIONS TO THE NUCLEAR MATRIX

The nucleus of a eukaryotic cell contains both structural and functional elements that contribute to the controlled operation of the cell. In this context, functional components refers to those nuclear constituents that perform metabolic activities such as DNA replication and RNA transcription. Structural nuclear components, designated nuclear matrix, organize the DNA into loops or domains and appear to provide a framework for nuclear DNA organization. However, the boundary between structural and functional components is not clear cut as evinced by reports of associations between metabolic functions and the nuclear matrix. The studies reported here attempt to determine the relationship of another nuclear function, DNA repair, to the nuclear matrix.^ One objective of these studies was to study the initiation of DNA repair by directly measuring the UV-incision activities in human cells and determine the influence of various extractable nuclear components on these activities. The assay for incision activities required the development of a nuclear isolation protocol that produced nuclei with intact DNA; the conformation of the nuclear DNA and its physical characteristics in response to denaturing conditions were determined.^ The nuclei produced with this protocol were then used as substrates for endogenous UV-specific nuclease activities. The isolated nuclei were shown to contain activities that cause breaks in nuclear DNA in response to UV-irradiation. These UV-responsive activities were tightly associated with nuclear components, being unextractable with salt concentration of up to 0.6 M.^ The tight association of the incision activities with salt-extracted nuclei suggested that other repair function might also be associated with salt-stable components of the nucleus. The site of unscheduled DNA synthesis (UDS) was determined in salt-extracted nuclei (nucleoids) using autoradiography and fluorescent microscopy. UDS was found to occur in association with the nuclear matrix following low-doses (2.55 J/M('2)) of ultraviolet light, but the association became looser after higher doses of ultraviolet light (10-30 J/m('2)). ^

DNA incisions stimulate genetic instability of triplet repeat sequences related to human hereditary neurological diseases

The molecular mechanisms responsible for the expansion and deletion of trinucleotide repeat sequences (TRS) are the focus of our studies. Several hereditary neurological diseases including Huntington's disease, myotonic dystrophy, and fragile X syndrome are associated with the instability of TRS. Using the well defined and controllable model system of Escherichia coli, the influences of three types of DNA incisions on genetic instability of CTG•CAG repeats were studied: DNA double-strand breaks (DSB), single-strand nicks, and single-strand gaps. The DNA incisions were generated in pUC19 derivatives by in vitro cleavage with restriction endonucleases. The cleaved DNA was then transformed into E. coli parental and mutant strains. Double-strand breaks induced deletions throughout the TRS region in an orientation dependent manner relative to the origin of replication. The extent of instability was enhanced by the repeat length and sequence (CTG•CAG vs. CGG•CCG). Mutations in recA and recBC increased deletions, mutations in recF stabilized the TRS, whereas mutations in ruvA had no effect. DSB were repaired by intramolecular recombination, versus an intermolecular gene conversion or crossover mechanism. 30 nt gaps formed a distinct 30 nt deletion product, whereas single strand nicks and gaps of 15 nts did not induce expansions or deletions. Formation of this deletion product required the CTG•CAG repeats to be present in the single-stranded region and was stimulated by E. coli DNA ligase, but was not dependent upon the RecFOR pathway. Models are presented to explain the DSB induced instabilities and formation of the 30 nucleotide deletion product. In addition to the in vitro creation of DSBs, several attempts to generate this incision in vivo with the use of EcoR I restriction modification systems were conducted. ^

Elucidation of the role of 53BP1 in DNA double strand break (DSB) repair and tumor suppression

The protein p53 binding protein one (53BP1) was discovered in a yeast two-hybrid screen that used the DNA binding domain of p53 as bait. Cloning of full-length 53BP1 showed that this protein contains several protein domains which help make up the protein, which include two tandem BRCT domains and a amino-terminal serine/glutamine cluster domain (SCD). These are two protein domains are often seen in factors that are involved in the cellular response to DNA damage and control of cell cycle checkpoints and we hypothesize that 53BP1 is involved in the cellular response to DNA damage. In support of this hypothesis we observe that 53BP1 is phosphorylated and undergoes a dramatic nuclear re-localization in response to DNA damaging agents. 53BP1 also interacts with several factors that are important in the cellular response to DNA damage, such as the BRCA1 tumor suppressor, ATM and Rad3 related (ATR), and the phosphorylated version of the histone variant H2AX. Mice deficient in 53BP1 display increased sensitivity ionizing radiation (IR), a DNA damaging agent that introduces DNA double strand breaks (DSBs). In addition, 53BP1-deficient mice do not properly undergo the process of class switch recombination (CSR). We also observe that when a defect in 53BP1 is combined with a defect in p53; the resulting mice have an increased rate of formation of spontaneous tumors, notably the formation of B and T lineage lymphomas. The T lineage tumors arise by two distinct mechanisms: one driven by defects in cell cycle regulation and a second driven by defects in the ability to repair DNA DSBs. The B lineage tumors arise by the inability to repair DNA damage and over-expression of the oncogene c-myc. ^ With these observations, we conclude that not only does 53BP1 function in the cellular response to DNA damage, but it also works in concert with p53 to suppress tumor formation. ^

The molecular mechanisms of sensing nucleoside analogue-induced DNA damage

Nucleoside analogues are antimetabolites effective in the treatment of a wide variety of solid tumors and hematological malignancies. Upon being metabolized to their active triphosphate form, these agents are incorporated into DNA during replication or excision repair synthesis. Because DNA polymerases have a greatly decreased affinity for primers terminated by most nucleoside analogues, their incorporation causes stalling of replication forks. The molecular mechanisms that recognize blocked replication may contribute to drug resistance but have not yet been elucidated. Here, several molecules involved in sensing nucleoside analogue-induced stalled replication forks have been identified and examined for their contribution to drug resistance. ^ The phosphorylation of the DNA damage sensor, H2AX, was characterized in response to nucleoside analogues and found to be dependent on both time and drug concentration. This response was most evident in the S-phase fraction and was associated with an inhibition of DNA synthesis, S-phase accumulation, and activation of the S-phase checkpoint pathway (Chk1-Cdc25A-Cdk2). Exposure of the Chk1 inhibitor, 7-hydroxystaurosporine (UCN-01), to cultures previously treated with nucleoside analogues caused increased apoptosis, clonogenic death, and a further log-order increase in H2AX phosphorylation, suggesting enhanced DNA damage. Ataxia-telangiectasia mutated (ATM) has been identified as a key DNA damage signaling kinase for initiating cell cycle arrest, DNA repair, and apoptosis while the Mre11-Rad50-Nbs1 (MRN) complex is known for its functions in double-strand break repair. Activated ATM and the MRN complex formed distinct nuclear foci that colocalized with phosphorylated H2AX after inhibition of DNA synthesis by the nucleoside analogues, gemcitabine, ara-C, and troxacitabine. Since double-strand breaks were undetectable, this response was likely due to stalling of replication forks. A similar DNA damage response was observed in human lymphocytes after exposure to ionizing radiation and in acute myelogenous leukemia blasts during therapy with the ara-C prodrug, CP-4055. Deficiencies in ATM, Mre11, and Rad50 led to a two- to five-fold increase in gemcitabine sensitivity, suggesting that these molecules contribute to drug resistance. Based on these results, a model is proposed for the sensing of nucleoside analogue-induced stalled replication forks that includes H2AX, ATM, and the Mre11-Rad50-Nbs1 complex. ^

E2F1's role in ultraviolet-induced DNA damage response

The E2F1 transcription factor is a well-known regulator of cell proliferation and apoptosis, but its role in the DNA damage response is less clear. It has been shown that E2F1 becomes stabilized in response to DNA double strand breaks (DSBs) and accumulates at sites of DSBs. This process requires ATM kinase and serine 31 phosphorylation, which provides a binding site for TopBp1. However, the role of E2F1 at sites of DNA damage is not clear. We expanded the study of E2F1's role in the DNA damage response by exploring its functions in ultraviolet (UV) induced DNA damage, and identified that E2F1 promotes DNA repair and cell survival. To further investigate the mechanisms underlying our findings, we examined the possibility for direct involvement of E2F1 in DNA repair. We found that E2F1 localizes to sites of UV irradiation-induced DNA damage dependent on the ATR kinase and serine 31 of E2F1. E2F1 also associates with the GCN5 histone acetyltransferase in response to UV irradiation and recruits GCN5 to sites of DNA damage. This correlates with an increase in histone H3 lysine 9 (H3K9) acetylation and chromatin relaxation. In the absence of E2F1 or GCN5, nucleotide excision repair (NER) proteins do not efficiently localize to sites of UV damage and DNA repair is impaired. E2F1 mutants unable to bind DNA or activate transcription retain the ability to stimulate NER. These findings demonstrate a non-transcriptional role for E2F1 in DNA repair involving GCN5-mediated H3K9 acetylation and increased accessibility to the NER machinery. ^

Understanding the roles of Non-homologous end joining and p53 after DNA damage

The inability to maintain genomic stability and control proliferation are hallmarks of many cancers, which become exacerbated in the presence of unrepaired DNA damage. Such genotoxic stresses trigger the p53 tumor suppressor network to activate transient cell cycle arrest allowing for DNA repair; if the damage is excessive or irreparable, apoptosis or cellular senescence is triggered. One of the major DNA repair pathway that mends DNA double strand breaks is non-homologous end joining (NHEJ). Abrogating the NHEJ pathway leads to an accumulation of DNA damage in the lymphoid system that triggers p53-mediated apoptosis; complete deletion of p53 in this system leads to aggressive lymphomagenesis. Therefore, to study the effect of p53-dependent cell cycle arrest, we utilized a hypomorphic, separation-of-function mutant, p53p/p, which completely abrogates apoptosis yet retains partial cell cycle arrest ability. We crossed DNA ligase IV deficiency, a downstream ligase crucial in mending breaks during NHEJ, into the p53p/p background (Lig4-/-p53p/p). The accumulation of DNA damage activated the p53/p21 axis to trigger cellular senescence in developing lymphoid cells, which absolutely suppressed tumorigenesis. Interestingly, these mice progressively succumb to severe diabetes. Mechanistic analysis revealed that spontaneous DNA damage accumulated in the pancreatic b-cells, a unique subset of endocrine cells solely responsible for insulin production to regulate glucose homeostasis. The genesis of adult b-cells predominantly occurs through self-replication, therefore modulating cellular proliferation is an essential component for renewal. The progressive accumulation of DNA damage, caused by Lig4-/-, activated p53/p21-dependent cellular senescence in mutant pancreatic b-cells that lead to islet involution. Insulin levels subsequently decreased, deregulating glucose homeostasis driving overt diabetes. Our Lig4-/-p53p/p model aptly depicts the dichotomous role of cellular senescence—in the lymphoid system prevents tumorigenesis yet in the endocrine system leads to the decrease of insulin-producing cells causing diabetes. To further delineate the function of NHEJ in pancreatic b-cells, we analyzed mice deficient in another component of the NHEJ pathway, Ku70. Although most notable for its role in DNA damage recognition and repair within the NHEJ pathway, Ku70 has NHEJ-independent functions in telomere maintenance, apoptosis, and transcriptional regulation/repression. To our surprise, Ku70-/-p53p/p mutant mice displayed a stark increase in b-cell proliferation, resulting in islet expansion, heightened insulin levels and hypoglycemia. Augmented b-cell proliferation was accompanied with the stabilization of the canonical Wnt pathway, responsible for this phenotype. Interestingly, the progressive onset of cellular senescence prevented islet tumorigenesis. This study highlights Ku70 as an important modulator in not only maintaining genomic stability through NHEJ-dependent functions, but also reveals a novel NHEJ-independent function through regulation of pancreatic b-cell proliferation. Taken in aggregate, these studies underscore the importance for NHEJ to maintain genomic stability in b-cells as well as introduces a novel regulator for pancreatic b-cell proliferation.

Su(UR)ES: A gene suppressing DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster polytene chromosomes

A genetic locus suppressing DNA underreplication in intercalary heterochromatin (IH) and pericentric heterochromatin (PH) of the polytene chromosomes of Drosophila melanogaster salivary glands, has been described. Found in the In(1)scV2 strain, the mutation, designated as Su(UR)ES, was located on chromosome 3L at position 34.8 and cytologically mapped to region 68A3-B4. A cytological phenotype was observed in the salivary gland chromosomes of larvae homozygous and hemizygous for Su(UR)ES: (i) in the IH regions, that normally are incompletely polytenized and so they often break to form “weak points,” underreplication is suppressed, breaks and ectopic contacts disappear; (ii) the degree of polytenization in PH grows higher. That is why the regions in chromosome arm basements, normally β-heterochromatic, acquire a distinct banding pattern, i.e., become euchromatic by morphological criteria; (iii) an additional bulk of polytenized material arises between the arms of chromosome 3 to form a fragment with a typical banding pattern. Chromosome 2 PH reveals additional α-heterochromatin. Su(UR)ES does not affect the viability, fertility, or morphological characters of the imago, and has semidominant expression in the heterozygote and distinct maternal effect. The results obtained provide evidence that the processes leading to DNA underreplication in IH and PH are affected by the same genetic mechanism.

Highly conservative reciprocal translocations formed by apparent joining of exchanged DNA double-strand break ends

Chromosomal translocations induced by ionizing radiation and radiomimetic drugs are thought to arise by incorrect joining of DNA double-strand breaks. To dissect such misrepair events at a molecular level, large-scale, bleomycin-induced rearrangements in the aprt gene of Chinese hamster ovary D422 cells were mapped, the breakpoints were sequenced, and the original non-aprt parental sequences involved in each rearrangement were recovered from nonmutant cells. Of seven rearrangements characterized, six were reciprocal exchanges between aprt and unrelated sequences. Consistent with a mechanism involving joining of exchanged double-strand break ends, there was, in most cases, no homology between the two parental sequences, no overlap in sequences retained at the two newly formed junctions, and little or no loss of parental sequences (usually ≤2 bp) at the breakpoints. The breakpoints were strongly correlated (P < 0.0001) with expected sites of bleomycin-induced, double-strand breaks. Fluorescence in situ hybridization indicated that, in six of the mutants, the rearrangement was accompanied by a chromosomal translocation at the aprt locus, because upstream and downstream flanking sequences were detected on separate chromosomes. The results suggest that repair of free radical-mediated, double-strand breaks in confluence-arrested cells is effected by a conservative, homology-independent, end-joining pathway that does not involve single-strand intermediate and that misjoining of exchanged ends by this pathway can directly result in chromosomal translocations.

Requirement of poly(ADP-ribose) polymerase in recovery from DNA damage in mice and in cells

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+: poly(adenosine-diphosphate-d-ribosyl)-acceptor ADP-d-ribosyltransferase, EC 2.4.2.30] is a zinc-finger DNA-binding protein that detects specifically DNA strand breaks generated by genotoxic agents. To determine its biological function, we have inactivated both alleles by gene targeting in mice. Treatment of PARP−/− mice either by the alkylating agent N-methyl-N-nitrosourea (MNU) or by γ-irradiation revealed an extreme sensitivity and a high genomic instability to both agents. Following whole body γ-irradiation (8 Gy) mutant mice died rapidly from acute radiation toxicity to the small intestine. Mice-derived PARP−/− cells displayed a high sensitivity to MNU exposure: a G2/M arrest in mouse embryonic fibroblasts and a rapid apoptotic response and a p53 accumulation were observed in splenocytes. Altogether these results demonstrate that PARP is a survival factor playing an essential and positive role during DNA damage recovery.

Double strand breaks at the HIS2 recombination hot spot in Saccharomyces cerevisiae

Double strand breaks (DSBs) have been found at several meiotic recombination hot spots in Saccharomyces cerevisiae; more global studies have found that they occur at many places along several yeast chromosomes during meiosis. Indeed, the number of breaks found is consistent with the number of recombination events predicted from the genetic map. We have previously demonstrated that the HIS2 gene is a recombination hot spot, exhibiting a high frequency of gene conversion and associated crossing over. This paper shows that DSBs occur in meiosis at a site in the coding region and at a site downstream of the HIS2 gene and that the DSBs are dependent upon genes required for recombination. The frequency of DSBs at HIS2 increases when the gene conversion frequency is increased by alterations in the DNA around HIS2, and vice versa. A deletion that increases both DSBs and conversion can stimulate both when heterozygous; that is, it is semidominant and acts to stimulate DSBs in trans. These data are consistent with the view that homologous chromosomes associate with each other before the formation of the DSBs.