Secondary structure formation and DNA instability at fragile site FRA16B

Human chromosomal fragile sites are specific loci that are especially susceptible to DNA breakage following conditions of partial replication stress. They often are found in genes involved in tumorigenesis and map to over half of all known cancer-specific recurrent translocation breakpoints. While their molecular basis remains elusive, most fragile DNAs contain AT-rich flexibility islands predicted to form stable secondary structures. To understand the mechanism of fragile site instability, we examined the contribution of secondary structure formation to breakage at FRA16B. Here, we show that FRA16B forms an alternative DNA structure in vitro. During replication in human cells, FRA16B exhibited reduced replication efficiency and expansions and deletions, depending on replication orientation and distance from the origin. Furthermore, the examination of a FRA16B replication fork template demonstrated that the majority of the constructs contained DNA polymerase paused within the FRA16B sequence, and among the molecules, which completed DNA synthesis, 81% of them underwent fork reversal. These results strongly suggest that the secondary-structure-forming ability of FRA16B contributes to its fragility by stalling DNA replication, and this mechanism may be shared among other fragile DNAs.     

A linkage group with FRA16B (the fragile site at 16q22.1)

Summary Polymorphic DNA markers located in bands 16q13, 16q21 and 16q22 were examined for recombination with FRA16B, the fragile site at 16q22.100. A tight linkage cluster D16S10-FRA16B-D16S4-HP was established. There were no recombinants between D16S10 and D16S4, which flank FRA16B. The markers D16S10 and D16S4 are in close proximity on the genetic map and delineate a small chromosomal segment, which contains the distamycin A-inducible fragile site.     

129 The molecular mechanism of breakage at fragile site FRA16D

Replication stress induces physical breakage at discrete loci in chromosomes, which can be visualized on a metaphase chromosome spread. These common fragile sites (CFS) are conserved across species and are hotspots for sister chromatid recombination, viral integration, rearrangements, translocations, and deletions (Glover et al 2005). Despite multiple theories, the molecular mechanisms of CFS expression and genomic instability are still not well understood. The fragile site FRA16D is of special interest because it is the second most highly expressed fragile site and is located within the WWOX tumor suppressor gene. Previous data identified a polymorphic AT repeat within a FRA16D subregion called F1 that causes chromosome fragility and replication fork stalling in a yeast model (Zhang and Freudenreich 2007). Recently, we have found that breakage increases in an AT repeat length-dependent manner. Our results suggest that the AT repeat in the context of F1 forms a secondary structure, making the region more vulnerable to breakage.     

DNA structure and the Werner protein modulate human DNA polymerase delta-dependent replication dynamics within the common fragile site FRA16D

Common fragile sites (CFS) are chromosomal regions that exhibit instability during DNA replication stress. Although the mechanism of CFS expression has not been fully elucidated, one known feature is a severely delayed S-phase. We used an in vitro primer extension assay to examine the progression of DNA synthesis through various sequences within FRA16D by the replicative human DNA polymerases δ and α, and with human cell-free extracts. We found that specific cis-acting sequence elements perturb DNA elongation, causing inconsistent DNA synthesis rates between regions on the same strand and complementary strands. Pol δ was significantly inhibited in regions containing hairpins and microsatellites, [AT/TA]₂₄ and [A/T]_19–28, compared with a control region with minimal secondary structure. Pol δ processivity was enhanced by full length Werner Syndrome protein (WRN) and by WRN fragments containing either the helicase domain or DNA-binding C-terminal domain. In cell-free extracts, stalling was eliminated at smaller hairpins, but persisted in larger hairpins and microsatellites. Our data support a model whereby CFS expression during cellular stress is due to a combination of factors—density of specific DNA secondary-structures within a genomic region and asymmetric rates of strand synthesis.     

The rare human fragile site 16B

Susceptibility to scrapie is primarily controlled by polymorphisms in the ovine prion protein gene (PRNP). Here, we report a novel ovine exon three PRNP polymorphism (SNP G346C; P116), its association with the ovine ARQ allele (P116A136R154Q171), and two new genotypes (PARQ/ARR; PARQ/ARQ) for the St. Croix White (SCW) breed and a related composite (CMP) breed developed for meat production. The (P116) polymorphism occurs between the N-terminal cleavage site and the hydrophobic region of the ovine prion protein, a region which exhibits extreme conservation across mammalian taxa. The relatively high frequency (0.75) of resistant ARR alleles and the absence of ARQ alleles for the SCW ewes used as breeding stock for CMP resulted in significant genic differentiation (P = 0.0123; S.E. = 0.00113). Additionally, the majority of the SCW (66.7%) and CMP (65.4%) sampled possessed genotypes considered resistant or nearly resistant to scrapie and experimental BSE (bovine spongiform encephalopathy.     

The characterization of the common fragile site FRA16D and its involvement in multiple myeloma translocations

Fragile sites appear as breaks, gaps, or decondensations on metaphase chromosomes when cells are grown under specific culture conditions. The breaks are nonrandom, appearing in defined, conserved locations throughout the mammalian genome. Common fragile sites, as their name implies, are present in virtually all individuals. With three common fragile sites cloned, their mechanism of expression and the role, if any, they play in human disease are still unclear. We have assembled a BAC contig of >1 Mb across the second most active common fragile site, FRA16D (16q23.2). We fluorescently labeled these BACs and used them as probes on metaphases from aphidicolin-induced lymphocytes and demonstrated that FRA16D decondensation/breakage occurs over a region of at least 1 Mb. Thus, this is the largest common fragile site cloned to date. Microsatellite markers that map within FRA16D show a very high loss in prostate, breast, and ovarian tumors, indicating that loss within this fragile site may be important in the development or progression of these tumors. In addition, a common t(14q32;16q23) translocation is observed in up to 25% of all multiple myelomas (MM). We localized four of four such cloned t(14;16) MM breakpoints within the FRA16D region. This work further demonstrates that the common fragile sites may play an important role in cancer development.     

Characterization of the human common fragile site FRA2G

Common fragile sites are nonrandom loci that show gaps and breaks when cells are exposed to specific compounds. They are preferentially involved in recombination, chromosomal rearrangements, and foreign DNA integration. These sites have been suggested to play a role in chromosome instability observed in cancer. In this work we used a FISH-based approach to identify a BAC contig that spans the FRA2G fragile site located at the 2q31 region. Our observations indicate that a very fragile region spanning at least 450 kb is present within a large fragile region that extends over 1 Mb. At least seven genes are mapped in the fragile region. One of these seems to be a good candidate as a potential tumor suppressor gene impaired by the recurrent deletions observed at the 2q31 region in some neoplasms. In the fragile region, a considerable number of regions of high flexibility that may be related to the fragility are present.     

The neurobeachin gene spans the common fragile site FRA13A

Common fragile sites are normal constituents of chromosomal structure prone to chromosomal breakage. In humans, the cytogenetic locations of more than 80 common fragile sites are known. The DNA at 11 of them has been defined and characterized at the molecular level. According to the Genome Database, the common fragile site FRA13A maps to chromosome band 13q13.2. Here, we identify the precise genomic position of FRA13A, and characterize the genetic complexity of the fragile DNA sequence. We show that FRA13A breaks are limited to a 650 kb region within the neurobeachin (NBEA) gene, which genomically spans approximately 730 kb. NBEA encodes a neuron-specific multidomain protein implicated in membrane trafficking that is predominantly expressed in the brain and during development.     

Induction of distamycin A-inducible rare fragile sites and increased sister chromatid exchanges at the fragile site

Summary Expression of distamycin A-inducible rare fragile sites by AT-specific DNA-ligands was examined in lymphoblastoid cell lines derived from heterozygous carriers for the fra(8)(q24), fra(16)(pl2), and fra(16)(q22) sites. The sensitivity of fragile site expression to the inducers was different at these fragile sites. The expression of fra(8)(q24) was induced markedly by Hoechst 33258, but not by distamycin A or berenil. An increased expression of fra(16)(p12) was found following treatment with Hoechst 33258 or berenil, but not with distamycin A. At fra(16)(q22), distamycin A markedly induced the fragile site, but Hoechst 33258 and berenil did not. Since their response to the different inducers was similar to that found in cultured lymphocytes, lymphoblastoid cell lines appear to retain their inherent properties. Although BrdUrd alone did nto induce any fragile sites, concomitant treatment with BrdUrd plus the inducer was synergistically effective in inducing all the fragile sites. An increased frequency of sister chromatid exchanges was observed at fra(16)(p12) following simultaneous treatment with BrdUrd and berenil, mainly when the site was expressed as an isochromatid gap. Thus, the induced fra (16)(pl2) site is a hot spot for the formation of sister chromatid exchanges, as found in other reported fragile sites.     

CGG-repeat expansion in the DIP2B gene is associated with the fragile site FRA12A on chromosome 12q13.1

A high level of cytogenetic expression of the rare folate-sensitive fragile site FRA12A is significantly associated with mental retardation. Here, we identify an elongated polymorphic CGG repeat as the molecular basis of FRA12A. This repeat is in the 5' untranslated region of the gene DIP2B, which encodes a protein with a DMAP1-binding domain, which suggests a role in DNA methylation machinery. DIP2B mRNA levels were halved in two subjects with FRA12A with mental retardation in whom the repeat expansion was methylated. In two individuals without mental retardation but with an expanded and methylated repeat, DIP2B expression was reduced to approximately two-thirds of the values observed in controls. Interestingly, a carrier of an unmethylated CGG-repeat expansion showed increased levels of DIP2B mRNA, which suggests that the repeat elongation increases gene expression, as previously described for the fragile X-associated tremor/ataxia syndrome. These data suggest that deficiency of DIP2B, a brain-expressed gene, may mediate the neurocognitive problems associated with FRA12A.     

Human DNA ligase I efficiently seals nicks in nucleosomes

The access to DNA within nucleosomes is greatly restricted for most enzymes and trans-acting factors that bind DNA. We report here that human DNA ligase I, which carries out the final step of Okazaki fragment processing and of many DNA repair pathways, can access DNA that is wrapped about the surface of a nucleosome in vitro and carry out its enzymatic function with high efficiency. In addition, we find that ligase activity is not affected by the binding of linker histone (H1) but is greatly influenced by the disposition of the core histone tail domains. These results suggest that the window of opportunity for human DNA ligase I may extend well beyond the first stages of chromatin reassembly after DNA replication or repair.     

Matrix attachment region (MAR) properties and abnormal expansion of AT island minisatellites in FRA16B fragile sites in leukemic CEM cells

AT-rich minisatellites (AT islands) are sites of genomic instability in cancer cells and targets for extremely lethal AT-specific drugs, such as bizelesin. Here we investigated the AT islands in the FRA16B fragile site region for their possible roles in the organization of DNA on the nuclear matrix. The FRA16B AT island nominally spans ~3 kb of mostly >90% A/T DNA. In silico analysis indicates that this domain exhibits characteristics of nuclear matrix attachment regions (MARs): an exceptionally intense computed ‘MAR potential’ and profound duplex destabilization and flexibility. FRA16B repeats specifically bind to isolated nuclear matrices, which indicates their in vitro MAR function. This binding is several-fold greater than that of a known MAR in the c-myc gene. AT islands in fragile sites FRA16B and FRA16D are significantly more abundant in CEM cells that are hypersensitive to bizelesin compared to normal WI-38 cells. FRA16B overabundance in CEM is due to an ~10-fold expansion of FRA16B repeats. The expanded FRA16B minisatellites in CEM cells preferentially localize to the nuclear matrix-associated DNA indicating their in vivo MAR function. The unexpanded repeats in WI-38 cells localize to the loop DNA. The c-myc MAR is also matrix-associated in CEM cells while localizing to loop DNA in WI-38 cells. These results are the first to demonstrate that AT islands in fragile sites can function as MARs both in vitro and in vivo. The ability of FRA16B-mediated MAR sites to rearrange depending on the repeat expansion status could be relevant to both genomic instability of cancer cells and their sensitivity to AT-island targeting drugs.     

New polymorphic DNA marker close to the fragile site FRAXA

DNA from a human-hamster hybrid cell line, 908-K1B17, containing a small terminal portion of the long arm of the human X chromosome as well as the pericentric region of 19q was used as starting material for the isolation of an X-chromosome-specific DNA segment, RN1 (DXS369), which identifies a XmnI RFLP. Linkage analysis in fragile X families resulted in a maximum lod score of 15.3 at a recombination fraction of 0.05 between RN1 and fra(X). Analysis of recombinations around the fra(X) and distal to DXS105. Analysis of the marker content of hybrid cell line 908K1B17 suggests the localization of RN1 between DXS98 and fra(X). Heterozygosity of DXS369 is approximately 50%, which extends the diagnostic potential of RFLP analysis in fragile X families significantly.     

A novel minisatellite repeat expansion identified at FRA16B in a Japanese carrier

Previously, the allelic expansion of a 33-bp AT-rich minisatellite repeat has been reported to cause FRA16B, a distamycin A-inducible fragile site. Here, we identified a novel 35-bp minisatellite repeat at FRA16B in a Japanese carrier. The nucleotide sequence of the 35-bp minisatellite was highly AT-rich and nearly identical to the 33-bp one but with insertion of two nucleotides, thymine and adenine. The copy number of the AT-rich minisatellite was 21 in total in the carrier, while only a few copies of the 33-bp minisatellite were present in a non-carrier Japanese subject. These results suggest that the molecular mechanism involved in the allelic expansion of the minisatellite repeat in FRA16B recognizes both minisatellites, the 33-bp one and the 35-bp one, as an amplicon. These observations were different from the ones at folate-sensitive fragile sites, where the CCG triplet repeat was commonly involved in the allelic expansion. Although a slight reduction in AT content (95% > 90%) in the region of minisatellite expansion in the carrier subject was observed, both AT-content and length of the highly AT-rich region seem to play important roles in the cytogenetic expression of the distamycin A-inducible fragile site. In another normal subject, without fragile site expression, allelic expansion involving the 33-bp minisatellite was observed, and the length of the AT-rich DNA region was increased up to approximately 1000 bp. Since the length of the AT-rich minisatellite region was increased up to approximately 1,100-bp in the carrier subject, the threshold length for the cytogenetic expression of the AT-rich DNA region may be between about 1,000-bp and 1,100-bp.