Effective intra-S checkpoint responses to UVC in primary human melanocytes and melanoma cell lines.

The objective of this study was to assess potential functional attenuation or inactivation of the intra-S checkpoint during melanoma development. Proliferating cultures of skin melanocytes, fibroblasts, and melanoma cell lines were exposed to increasing fluences of UVC and intra-S checkpoint responses were quantified. Melanocytes displayed stereotypic intra-S checkpoint responses to UVC qualitatively and quantitatively equivalent to those previously demonstrated in skin fibroblasts. In comparison with fibroblasts, primary melanocytes displayed reduced UVC-induced inhibition of DNA strand growth and enhanced degradation of p21Waf1 after UVC, suggestive of enhanced bypass of UVC-induced DNA photoproducts. All nine melanoma cell lines examined, including those with activating mutations in BRAF or NRAS oncogenes, also displayed proficiency in activation of the intra-S checkpoint in response to UVC irradiation. The results indicate that bypass of oncogene-induced senescence during melanoma development was not associated with inactivation of the intra-S checkpoint response to UVC-induced DNA replication stress.

DNA damage checkpoint responses in the S phase of synchronized diploid human fibroblasts.

We investigated the hypothesis that the strength of the activation of the intra-S DNA damage checkpoint varies within the S phase. Synchronized diploid human fibroblasts were exposed to either 0 or 2.5 J m(-2) UVC in early, mid- and late-S phase. The endpoints measured were the following: (1) radio-resistant DNA synthesis (RDS), (2) induction of Chk1 phosphorylation, (3) initiation of new replicons and (4) length of replication tracks synthesized after irradiation. RDS analysis showed that global DNA synthesis was inhibited by approximately the same extent (30 ± 12%), regardless of when during S phase the fibroblasts were exposed to UVC. Western blot analysis revealed that the UVC-induced phosphorylation of checkpoint kinase 1 (Chk1) on serine 345 was high in early and mid S but 10-fold lower in late S. DNA fiber immunostaining studies indicated that the replication fork displacement rate decreased in irradiated cells at the three time points examined; however, replicon initiation was inhibited strongly in early and mid S, but this response was attenuated in late S. These results suggest that the intra-S checkpoint activated by UVC-induced DNA damage is not as robust toward the end of S phase in its inhibition of the latest firing origins in human fibroblasts.

Cyclobutane Pyrimidine Dimer Density as a Predictive Biomarker of the Biological Effects of Ultraviolet Radiation in Normal Human Fibroblast.

This study compared biological responses of normal human fibroblasts (NHF1) to three sources of ultraviolet radiation (UVR), emitting UVC wavelengths, UVB wavelengths, or a combination of UVA and UVB (solar simulator; emission spectrum, 94.3% UVA and 5.7% UVB). The endpoints measured were cytotoxicity, intra-S checkpoint activation, inhibition of DNA replication and mutagenicity. Results show that the magnitude of each response to the indicated radiation sources was best predicted by the density of DNA cyclobutane pyrimidine dimers (CPD). The density of 6-4 pyrimidine-pyrimidone photoproducts was highest in DNA from UVC-irradiated cells (14% of CPD) as compared to those exposed to UVB (11%) or UVA-UVB (7%). The solar simulator source, under the experimental conditions described here, did not induce the formation of 8-oxo-7,8-dihydroguanine in NHF1 above background levels. Taken together, these results suggest that CPD play a dominant role in DNA damage responses and highlight the importance of using endogenous biomarkers to compare and report biological effects induced by different sources of UVR.

Is activation of the intra-S checkpoint in human fibroblasts an important factor in protection against UV-induced mutagenesis?

The ATR/CHK1-dependent intra-S checkpoint inhibits replicon initiation and replication fork progression in response to DNA damage caused by UV (UV) radiation. It has been proposed that this signaling cascade protects against UV-induced mutations by reducing the probability that damaged DNA will be replicated before it can be repaired. Normal human fibroblasts (NHF) were depleted of ATR or CHK1, or treated with the CHK1 kinase inhibitor TCS2312, and the UV-induced mutation frequency at the HPRT locus was measured. Despite clear evidence of S-phase checkpoint abrogation, neither ATR/CHK1 depletion nor CHK1 inhibition caused an increase in the UV-induced HPRT mutation frequency. These results question the premise that the UV-induced intra-S checkpoint plays a prominent role in protecting against UV-induced mutagenesis.

Separation of intra-S checkpoint protein contributions to DNA replication fork protection and genomic stability in normal human fibroblasts.

The ATR-dependent intra-S checkpoint protects DNA replication forks undergoing replication stress. The checkpoint is enforced by ATR-dependent phosphorylation of CHK1, which are mediated by the TIMELESS-TIPIN complex and CLASPIN. Although loss of checkpoint proteins is associated with spontaneous chromosomal instability, few studies have examined the contribution of these proteins to unchallenged DNA metabolism in human cells that have not undergone carcinogenesis or crisis. Furthermore, the TIMELESS-TIPIN complex and CLASPIN may promote replication fork protection independently of CHK1 activation. Normal human fibroblasts (NHF) were depleted of ATR, CHK1, TIMELESS, TIPIN or CLASPIN and chromosomal aberrations, DNA synthesis, activation of the DNA damage response (DDR) and clonogenic survival were evaluated. This work demonstrates in NHF lines from two individuals that ATR and CHK1 promote chromosomal stability by different mechanisms that depletion of CHK1 produces phenotypes that resemble more closely the depletion of TIPIN or CLASPIN than the depletion of ATR, and that TIMELESS has a distinct contribution to suppression of chromosomal instability that is independent of its heterodimeric partner, TIPIN. Therefore, ATR, CHK1, TIMELESS-TIPIN and CLASPIN have functions for preservation of intrinsic chromosomal stability that is separate from their cooperation for activation of the intra-S checkpoint response to experimentally induced replication stress. These data reveal a complex and coordinated program of genome maintenance enforced by proteins known for their intra-S checkpoint function.

Assays of bypass replication of genotoxic lesions in cell-free extracts.

The in vitro replication assay described here measures bidirectional replication of a circular double- stranded DNA template upon initiation at the SV40 origin. It models a single eukaryotic replication unit (replicon) and recapitulates the biochemical steps involved in the catalysis of both leading and lagging strand synthesis during semiconservative DNA replication. Except for the SV40 large T antigen, all other proteins necessary for initiation and assembly of functional replication forks are provided by the cell-free extract. This assay can be used to demonstrate bypass replication of genotoxic lesions. It supports replication across a specific damaged site on the template DNA (i.e., translesion synthesis) by specialized DNA polymerases. This chapter illustrates the efficient translesion synthesis of UV-induced thymine dimers by DNA polymerase eta.

Analyzing DNA replication dynamics of genotoxin-treated cells using velocity sedimentation.

Following acquisition of DNA damage S-phase progression may potentially be affected via multiple mechanisms. For example DNA damage-activated signal transduction pathways negatively regulate the initiation of DNA synthesis at unfired origins of replication, a process termed the 'S-phase checkpoint' or the 'intra-S-phase checkpoint'. Additionally, many DNA lesions pose physical barriers to replication forks and therefore inhibit DNA synthesis directly by blocking the elongation of active replicons. Inhibition of DNA synthesis in response to DNA damage is commonly assayed by measuring incorporation of radiolabeled or halogenated nucleotides into bulk genomic DNA. However, these techniques do not distinguish between effects of DNA damage on initiation and elongation phases of DNA synthesis. The velocity sedimentation protocol described here allows investigators to determine the effects of DNA damage on initiation and elongation events. This technique involves labeling replicating DNA with (3)H-thymidine, then analyzing the size distribution of labeled ssDNAs based on their differential density sedimentation profiles after centrifugation through alkaline sucrose gradients. Determining the relative abundance and growth rates of small nascent ssDNAs provides an index of initiation and elongation events, respectively. Therefore, analysis of replication dynamics using velocity sedimentation provides a potentially valuable tool for assaying S-phase checkpoints as well as other aspects of DNA replication.

Timeless functions independently of the Tim-Tipin complex to promote sister chromatid cohesion in normal human fibroblasts.

The Timeless-Tipin complex and Claspin are mediators of the ATR-dependent activation of Chk1 in the intra-S checkpoint response to stalled DNA replication forks. Tim-Tipin and Claspin also contribute to sister chromatid cohesion (SCC) in various organisms, likely through a replication-coupled process. Some models of the establishment of SCC posit that interactions between cohesin rings and replisomes could result in physiological replication stress requiring fork stabilization. The contributions of Timeless, Tipin, Claspin, Chk1 and ATR to SCC were investigated in genetically stable, human diploid fibroblast cell lines. Whereas Timeless, Tipin and Claspin showed similar contributions to UVC-induced activation of Chk1, siRNA-mediated knockdown of Timeless induced a 100-fold increase in sister chromatid discohesion, whereas the inductive effects of knocking down Tipin, Claspin and ATR were 4-20-fold. Knockdown of Chk1 did not significantly affect SCC. Consistent findings were obtained in two independently derived human diploid fibroblast lines and support a conclusion that SCC in human cells is strongly dependent on Timeless but independent of Chk1. Furthermore, the 10-fold difference in discohesion observed when depleting Timeless versus Tipin indicates that Timeless has a function in SCC that is independent of the Tim-Tipin complex, even though the abundance of Timeless is reduced when Tipin is targeted for depletion. A better understanding of how Timeless, Tipin and Claspin promote SCC will elucidate non-checkpoint functions of these proteins at DNA replication forks and inform models of the establishment of SCC.

Revised genetic requirements for the decatenation G2 checkpoint: the role of ATM.

The decatenation G2 checkpoint is proposed to delay cellular progression from G2 into mitosis when intertwined daughter chromatids are insufficiently decatenated. Previous studies indicated that the ATM- and Rad3-related (ATR) checkpoint kinase, but not the ataxia telangiectasia-mutated (ATM) kinase, was required for decatenation G2 checkpoint function. Here, we show that the method used to quantify decatenation G2 checkpoint function can influence the identification of genetic requirements for the checkpoint. Normal human diploid fibroblast (NHDF) lines responded to the topoisomerase II (topo II) catalytic inhibitor ICRF-193 with a stringent G2 arrest and a reduction in the mitotic index. While siRNA-mediated depletion of ATR and CHEK1 increased the mitotic index in ICRF-193 treated NHDF lines, depletion of these proteins did not affect the mitotic entry rate, indicating that the decatenation G2 checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G2 checkpoint, but may influence mitotic exit after inhibition of topo II. A re-evaluation of ataxia telangiectasia (AT) cell lines using the mitotic entry assay indicated that ATM was required for the decatenation G2 checkpoint. Three NHDF cell lines responded to ICRF-193 with a mean 98% inhibition of the mitotic entry rate. Examination of the mitotic entry rates in AT fibroblasts upon treatment with ICRF-193 revealed a significantly attenuated decatenation G2 checkpoint response, with a mean 59% inhibition of the mitotic entry rate. In addition, a normal lymphoblastoid line exhibited a 95% inhibition of the mitotic entry rate after incubation with ICRF-193, whereas two AT lymphoblastoid lines displayed only 36% and 20% inhibition of the mitotic entry rate. Stable depletion of ATM in normal human fibroblasts with short hairpin RNA also attenuated decatenation G2 checkpoint function by an average of 40%. Western immunoblot analysis demonstrated that treatment with ICRF-193 induced ATM autophosphorylation and ATM-dependent phosphorylation of Ser15-p53 and Thr68 in Chk2, but no appreciable phosphorylation of Ser139-H2AX or Ser345-Chk1. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G2 arrest independently of DNA damage.

Tipin-replication protein A interaction mediates Chk1 phosphorylation by ATR in response to genotoxic stress.

Mammalian Timeless is a multifunctional protein that performs essential roles in the circadian clock, chromosome cohesion, DNA replication fork protection, and DNA replication/DNA damage checkpoint pathways. The human Timeless exists in a tight complex with a smaller protein called Tipin (Timeless-interacting protein). Here we investigated the mechanism by which the Timeless-Tipin complex functions as a mediator in the ATR-Chk1 DNA damage checkpoint pathway. We find that the Timeless-Tipin complex specifically mediates Chk1 phosphorylation by ATR in response to DNA damage and replication stress through interaction of Tipin with the 34-kDa subunit of replication protein A (RPA). The Tipin-RPA interaction stabilizes Timeless-Tipin and Tipin-Claspin complexes on RPA-coated ssDNA and in doing so promotes Claspin-mediated phosphorylation of Chk1 by ATR. Our results therefore indicate that RPA-covered ssDNA not only supports recruitment and activation of ATR but also, through Tipin and Claspin, it plays an important role in the action of ATR on its critical downstream target Chk1.

Human papilloma virus type16 E6 deregulates CHK1 and sensitizes human fibroblasts to environmental carcinogens independently of its effect on p53.

After treatment with ultraviolet radiation (UV), human fibroblasts that express the HPV type 16 E6 oncoprotein display defects in repair of cyclobutane pyrimidine dimers, hypersensitivity to inactivation of clonogenic survival and an inability to sustain DNA replication. To determine whether these effects are specific to depletion of p53 or inactivation of its function, fibroblast lines were constructed with ectopic expression of a dominant-negative p53 allele (p53-H179Q) to inactivate function or a short-hairpin RNA (p53-RNAi) to deplete expression of p53. Only the expression of HPV16E6 sensitized fibroblasts to UV or the chemical carcinogen, benzo[a]pyrene diolepoxide I (BPDE). Carcinogen-treated cells expressing p53-H179Q or p53-RNAi were resistant to inactivation of colony formation and did not suffer replication arrest. CHK1 is a key checkpoint kinase in the response to carcinogen-induced DNA damage. Control and p53-RNAi-expressing fibroblasts displayed phosphorylation of Ser345 on CHK1 45-120 min after carcinogen treatment with a return to near baseline phosphorylation by 6 h after treatment. HPV16E6-expressing fibroblasts displayed enhanced and sustained phosphorylation of CHK1. This was associated with enhanced phosphorylation of Thr68 on CHK2 and Ser139 on H2AX, both markers of severe replication stress and DNA double strand breaks. Incubation with the phosphatase inhibitor okadaic acid produced more phosphorylation of CHK1 in UV-treated HPV16E6-expressing cells than in p53-H179Q-expressing cells suggesting that HPV16E6 may interfere with the recovery of coupled DNA replication at replication forks that are stalled at [6-4]pyrimidine-pyrimidone photoproducts and BPDE-DNA adducts. The results indicate that HPV16E6 targets a protein or proteins other than p53 to deregulate the activity of CHK1 in carcinogen-damaged cells.

Origin licensing and p53 status regulate Cdk2 activity during G(1).

Origins of DNA replication are licensed through the assembly of a chromatin-bound prereplication complex. Multiple regulatory mechanisms block new prereplication complex assembly after the G(1)/S transition to prevent rereplication. The strict inhibition of licensing after the G(1)/S transition means that all origins used in S phase must have been licensed in the preceding G(1). Nevertheless mechanisms that coordinate S phase entry with the completion of origin licensing are still poorly understood. We demonstrate that depletion of either of two essential licensing factors, Cdc6 or Cdt1, in normal human fibroblasts induces a G(1) arrest accompanied by inhibition of cyclin E/Cdk2 activity and hypophosphorylation of Rb. The Cdk2 inhibition is attributed to a reduction in the essential activating phosphorylation of T160 and an associated delay in Cdk2 nuclear accumulation. In contrast, licensing inhibition in the HeLa or U2OS cancer cell lines failed to regulate Cdk2 or Rb phosphorylation, and these cells died by apoptosis. Co-depletion of Cdc6 and p53 in normal cells restored Cdk2 activation and Rb phosphorylation, permitting them to enter S phase with a reduced rate of replication and also to accumulate markers of DNA damage. These results demonstrate dependence on origin licensing for multiple events required for G(1) progression, and suggest a mechanism to prevent premature S phase entry that functions in normal cells but not in p53-deficient cells.

DNA replication and the GINS complex: localization on extended chromatin fibers.

BACKGROUND: The GINS complex is thought to be essential for the processes of initiation and elongation of DNA replication. This complex contains four subunits, one of which (Psf1) is proposed to bind to both chromatin and DNA replication-associated proteins. To date there have been no microscopic analyses to evaluate the chromatin distribution of this complex. Here, we show the organization of GINS complexes on extended chromatin fibers in relation to sites of DNA replication and replication-associated proteins. RESULTS: Using immunofluorescence microscopy we were able to visualize ORC1, ORC2, PCNA, and GINS complex proteins Psf1 and Psf2 bound to extended chromatin fibers. We were also able to detect these proteins concurrently with the visualization of tracks of recently replicated DNA where EdU, a thymidine analog, was incorporated. This allowed us to assess the chromatin association of proteins of interest in relation to the process of DNA replication. ORC and GINS proteins were found on chromatin fibers before replication could be detected. These proteins were also associated with newly replicated DNA in bead-like structures. Additionally, GINS proteins co-localized with PCNA at sites of active replication. CONCLUSION: In agreement with its proposed role in the initiation of DNA replication, GINS proteins associated with chromatin near sites of ORC binding that were devoid of EdU (absence of DNA replication). The association of GINS proteins with PCNA was consistent with a role in the process of elongation. Additionally, the large size of our chromatin fibers (up to approximately 7 Mb) allowed for a more expansive analysis of the distance between active replicons than previously reported.

Ubiquitylation of proliferating cell nuclear antigen and recruitment of human DNA polymerase eta.

This study investigated the requirement for ubiquitylation of PCNA at lysine 164 during polymerase eta-dependent translesion synthesis (TLS) of site-specific cis-syn cyclobutane thymine dimers (T (wedge)T). The in vitro assay recapitulated origin-dependent initiation, fork assembly, and semiconservative, bidirectional replication of double-stranded circular DNA substrates. A phosphocellulose column was used to fractionate HeLa cell extracts into two fractions; flow-through column fraction I (CFI) contained endogenous PCNA, RPA, ubiquitin-activating enzyme E1, and ubiquitin conjugase Rad6, and eluted column fraction II (CFII) included pol delta, pol eta, and RFC. CFII supplemented with purified recombinant RPA and PCNA (wild type or K164R, in which lysine was replaced with arginine) was competent for DNA replication and TLS. K164R-PCNA complemented CFII for these activities to the same extent and efficiency as wild-type PCNA. CFII mixed with CFI (endogenous PCNA, E1, Rad6) exhibited enhanced DNA replication activity, but the same TLS efficiency determined with the purified proteins. These results demonstrate that PCNA ubiquitylation at K164 of PCNA is not required in vitro for pol eta to gain access to replication complexes at forks stalled by T (wedge)T and to catalyze TLS across this dimer.

Defective cell cycle checkpoint functions in melanoma are associated with altered patterns of gene expression.

Defects in DNA damage responses may underlie genetic instability and malignant progression in melanoma. Cultures of normal human melanocytes (NHMs) and melanoma lines were analyzed to determine whether global patterns of gene expression could predict the efficacy of DNA damage cell cycle checkpoints that arrest growth and suppress genetic instability. NHMs displayed effective G1 and G2 checkpoint responses to ionizing radiation-induced DNA damage. A majority of melanoma cell lines (11/16) displayed significant quantitative defects in one or both checkpoints. Melanomas with B-RAF mutations as a class displayed a significant defect in DNA damage G2 checkpoint function. In contrast the epithelial-like subtype of melanomas with wild-type N-RAS and B-RAF alleles displayed an effective G2 checkpoint but a significant defect in G1 checkpoint function. RNA expression profiling revealed that melanoma lines with defects in the DNA damage G1 checkpoint displayed reduced expression of p53 transcriptional targets, such as CDKN1A and DDB2, and enhanced expression of proliferation-associated genes, such as CDC7 and GEMININ. A Bayesian analysis tool was more accurate than significance analysis of microarrays for predicting checkpoint function using a leave-one-out method. The results suggest that defects in DNA damage checkpoints may be recognized in melanomas through analysis of gene expression.

Early replication and the apoptotic pathway.

In higher eukaryotes there is a link between time of replication and transcription. It is generally accepted that genes that are actively transcribed are replicated in the first half of S phase while inactive genes replicate in the second half of S phase. We have recently reported that in normal human fibroblasts there are some functionally related genes that replicate at the same time in S phase. This had been previously reported for functionally related genes that are located in clusters, for example the alpha- and beta-globin complexes. We have shown, however, that this also occurs with some functionally related genes that are not organized in a cluster, but rather are distributed throughout the genome. For example, using GOstat analysis of data from our and other groups, we found an overrepresentation of genes involved in the apoptotic process among sequences that are replicated very early (approximately in the first hour of S phase) in both fibroblasts and lymphoblastoid cells. This finding leads us to question how and why the replication of genes in the apoptotic pathway is temporally organized in this manner. Here we discuss the possible explanations and implications of this observation.

The human Tim/Tipin complex coordinates an Intra-S checkpoint response to UV that slows replication fork displacement.

UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks, ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin, and Timeless (Tim). Murine Tim recently was shown to form a complex with Tim-interacting protein (Tipin), and a similar complex was shown to exist in human cells. Knockdown of Tipin using small interfering RNA reduced the expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with replication protein A (RPA) and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immunofluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m(2) UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. The depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of the control. In contrast, Tipin depletion had little or no effect on fork progression in unirradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA and, in UV-damaged cells, Tipin slows DNA chain elongation in active replicons.

Cdc7-Dbf4 and the human S checkpoint response to UVC.

The S checkpoint response to ultraviolet radiation (UVC) that inhibits replicon initiation is dependent on the ATR and Chk1 kinases. Downstream effectors of this response, however, are not well characterized. Data reported here eliminated Cdc25A degradation and inhibition of Cdk2-cyclin E as intrinsic components of the UVC-induced pathway of inhibition of replicon initiation in human cells. A sublethal dose of UVC (1 J/m(2)), which selectively inhibits replicon initiation by 50%, failed to reduce the amount of Cdc25A protein or decrease Cdk2-cyclin E kinase activity. Cdc25A degradation was observed after irradiation with cytotoxic fluences of UVC, suggesting that severe inhibition of DNA chain elongation and activation of the replication checkpoint might be responsible for the UVC-induced degradation of Cdc25A. Another proposed effector of the S checkpoint is the Cdc7-Dbf4 complex. Dbf4 interacted weakly with Chk1 in vivo but was recognized as a substrate for Chk1-dependent phosphorylation in vitro. FLAG-Dbf4 formed complexes with endogenous Cdc7, and this interaction was stable in UVC-irradiated HeLa cells. Overexpression of FLAG- or Myc-tagged Dbf4 abrogated the S checkpoint response to UVC but not ionizing radiation. These findings implicate a Dbf4-dependent kinase as a possible target of the ATR- and Chk1-dependent S checkpoint response to UVC.

Mapping of an origin of DNA replication in the promoter of fragile X gene FMR1.

An origin of bidirectional DNA replication was mapped to the promoter of the FMR1 gene in human chromosome Xq27.3, which has been linked to the fragile X syndrome. This origin is adjacent to a CpG island and overlaps the site of expansion of the triplet repeat (CGG) at the fragile X instability site, FRAXA. The promoter region of FMR2 in the FRAXE site (approximately 600 kb away, in chromosome band Xq28) also includes an origin of replication, as previously described [Chastain II, P.D., Cohen, S.M., Brylawski, B.P., Cordeiro-Stone, M., Kaufman, D.G., 2006. A late origin of DNA replication in the trinucleotide repeat region of the human FMR2 gene. Cell Cycle 5, 869-872]. FMR1 transcripts were detected in foreskin and male fetal lung fibroblasts, while FMR2 transcripts were not. However, both FMR1 and FMR2 were found to replicate late in S phase (approximately 6 h into the S phase of normal human fibroblasts). The position of the origin of replication relative to the CGG repeat, and perhaps the late replication of these genes, might be important factors in the susceptibility to triplet repeat amplification at the FRAXA and FRAXE sites.