FANCD2 promotes mitotic rescue from transcription-mediated replication stress in SETX-deficient cancer cells

Replication stress (RS) is a leading cause of genome instability and cancer development. A substantial source of endogenous RS originates from the encounter between the transcription and replication machineries operating on the same DNA template. This occurs predominantly under specific contexts, such as oncogene activation, metabolic stress, or a deficiency in proteins that specifically act to prevent or resolve those transcription-replication conflicts (TRCs). One such protein is Senataxin (SETX), an RNA:DNA helicase involved in resolution of TRCs and R-loops. Here we identify a synthetic lethal interaction between SETX and proteins of the Fanconi anemia (FA) pathway. Depletion of SETX induces spontaneous under-replication and chromosome fragility due to active transcription and R-loops that persist in mitosis. These fragile loci are targeted by the Fanconi anemia protein, FANCD2, to facilitate the resolution of under-replicated DNA, thus preventing chromosome mis-segregation and allowing cells to proliferate. Mechanistically, we show that FANCD2 promotes mitotic DNA synthesis that is dependent on XPF and MUS81 endonucleases. Importantly, co-depleting FANCD2 together with SETX impairs cancer cell proliferation, without significantly affecting non-cancerous cells. Therefore, we uncovered a synthetic lethality between SETX and FA proteins for tolerance of transcription-mediated RS that may be exploited for cancer therapy

THE EFFECT OF CAFFEINE ON DAPI-INDUCIBLE FRAGILE SITES

DAPI is a non-intercalating compound which binds specifically to the AT bases of DNA. When leukocytes are grown in complete medium (RPMI 1640) DAPI induces the expression of three fragile sites on human chromosomes and if the medium is deficient in folic acid and thymidine (199M) it induces 19 fragile sites. Caffeine has been found by different authors to considerably enhance the expression of chromosome breaks which have been produced by other agents. When it is added to the complete medium after DAPI, it elicits almost all the sites that DAPI only induces in incomplete medium. When caffeine is added after DA-PI to incomplete medium, it does not significantly or unidirectionally modify the capacity of the two subjects examined to elicit fragile sites. The analysis of these results, when correlated with that of the mitotic index, reveals a different sensitivity of the two subjects to the combined DAPI-caffeine treatment. The results are quite compatible with the hypothesis that the DAPI-induced fragile sites are DNA regions which are not accurately replicated during the S phase

Correction of the Wrong Name of a Fragile Site Associated to the DMD Gene

[No abstract available

SYNERGISTIC EFFECT OF DAPI AND THYMIDYLATE STRESS CONDITIONS ON THE INDUCTION OF COMMON FRAGILE SITES

When supplied to human leukocytes grown in complete medium (RPMI 1640), DAPI, a nonintercalating compound specific for the AT bases of DNA, induces the appearance of three common fragile sites (CFRA) mapped at 1q42, 2q31, and 7p22. The same treatment with DAPI in a medium deficient in folic acid and thymidine (199 M) considerably increases the expression of these sites and induces the appearance of a further 16 CFRA sites at 1q24, 2p25, 4p16, 4q25, 5p15.3, 6p21.3, 6p25, 6q13, 9p24, 16p13.3, 16q23, 17q21, 18q23, 20q13.1, 21q21, and Xq28. The results point to the existence of a synergism between DAPI and thymidylate-stress culture conditions in inducing site-specific chromosome damage. The results also agree with the hypothesis that DAPI-induced CFRA sites are DNA late-replicating chromosomal areas rich in AT bases

DAPI-INDUCIBLE COMMON FRAGILE SITES

DAPI, a compound specific for the AT bases of DNA, causes gaps and breaks in three human chromosome sites, at the 1q41-1q42 interface, 2q31, and 7p22. It also induces undercondensation of a chromosome site at the 13q21-13q22 interface. The first three sites have the characteristics of 'common fragile sites' and are present as gaps and breaks on the chromosomes of seven individuals

Inter- and intra-chromosomal distribution of chromatid breaks induced by X-rays during G2 in human lymphocytes

Cultures of blood from healthy adults were irradiated 48 h after stimulation with 240 R of X-rays and fixed after various time intervals (0-2 h, 2-4 h, 4-6 h). 3HTdR was added to several cultures after irradiation. Mitotic and labelling indices were used to distinguish between two cell samples inside the irradiated G2 population: D - cells reaching mitosis without mitotic delay and a high frequency of chromatic breaks and D + cells with mitotic delay and which, during the delay, repair most of the damage produced. After R banding 450 chromatid deletions were located in each of the two cell samples. The D + cells showed a higher frequency of breaks than the D - cells with decreasing chromosome size, in the telomeric and centromeric region and in the junction between the R + and R - bands. These results can be interpreted as indicative of a non-random distribution of repair processes both between and within chromosomes. © 1985

Breakages at common fragile sites set boundaries of amplified regions in two leukemia cell lines K562 - Molecular characterization of FRA2H and localization of a new CFS FRA2S

Genome amplification is often observed in human tumors The breakage-fusion-bridge (BFB) cycle is the mechanism that often underlies duplicated regions Some research has indicated common fragile sites (CFS) as possible sites of chromosome breakages at the on gin of BFB cycles Here we searched two human genome regions known as amplification hot spots for any DNA copy number amplifications by analyzing 21 cancer cell lines to investigate the relationship between genomic fragility and amplification We identified a duplicated region on a chromosomes der(2) present in the karyotype of two analysed leukemia cell lines K562 The two duplicated regions are organized into large palindromes which suggests that one BFB cycle has occurred Our findings show that the three breakpoints are localized in the sequence of three CFSs FRA2H (2q32 1-q32 2) which here has been characterized molecularly FRA2S (2q22 3-q23 3) a newly localized aphidicolin inducible CFS and FRA2G (204 3-q31) (C) 2010 Elsevier Ireland Ltd All rights reserve

Longitudinal differentiation of chromosomes of Asellus aquaticus (Crust. Isop.) by in situ nick translation using restriction enzymes and DNase I

Asellus aquaticus is an isopod crustacean whose chromosomes cannot be differentiated by G- or R-banding techniques. In this work, we have obtained a longitudinal differentiation of these chromosomes by in situ nick translation using restriction enzymes (HaeIII, DraI and BamHI) and DNase I digestions. The four nucleases, with different efficiencies, have produced similar labelling patterns. Staining with DAPI, Giemsa and chromomycin A(3) reveals that the DNA of the nick-translated regions is generally more resistant to extraction from the chromosome. The results obtained on the heteromorphic sex chromosome pair observed in about a quarter of the males of a natural population allow several hypotheses to be advanced on the nature and origin of chromosome dimorphism

Sex chromosome differentiation revealed by comparative genomic hybridization.

In this work, genomic in-situ hybridization (GISH) was used to study the sex chromosome molecular differentiation on chromosomes of male and female individuals of the isopod crustacean Asellus aquaticus. As a composite hybridization probe, we contemporaneously used male and female whole genomic DNA differently labelled in the presence of an excess of unlabelled DNA of the female homogametic sex. The karyotype of A. aquaticus normally displays eight homomorphic chromosome pairs, but a heteromorphic sex chromosome pair is present in about a quarter of the males of a natural population previously identi¢ed by us. GISH did not reveal any sex chromosome molecular differentiation on the male and female homomorphic sex chromosome pair, and the karyotypes of these individuals were equally labelled by the male- and female-derived probe, while the heteromorphic Y chromosome showed a differentially labelled region only with the male-derived probe. This region evidently contains male-speci¢c sequences but, because no similar hybridized region is observed on the male homomorphic chromosome pair, they are probably not important for sex determination but represent a molecular differentiation acquired from the Y chromosome. I.F.: 3.2