Sticky DNA, a Long GAA·GAA·TTC Triplex That Is Formed Intramolecularly, in the Sequence of Intron 1 of the Frataxin Gene

Friedreich's ataxia is caused by the massive expansion of GAA.TTC repeats in intron 1 of the frataxin (X25) gene. Our prior investigations showed that long GAA.TTC repeats formed very stable triplex structures which caused two repeat tracts to adhere to each other (sticky DNA). This process was dependent on negative supercoiling and the presence of divalent metal ions. Herein, we have investigated the formation of sticky DNA from plasmid monomers and dimers; sticky DNA is formed only when two tracts of sufficiently long (GAA.TTC)(n) (n = 59-270) are present in a single plasmid DNA and are in the direct repeat orientation. If the inserts are in the indirect (inverted) repeat orientation, no sticky DNA was observed. Furthermore, kinetic studies support the intramolecular nature of sticky DNA formation. Electron microscopy investigations also provide strong data for sticky DNA as a single long triplex. Hence, these results give new insights into our understanding of the capacity of sticky DNA to inhibit transcription and thereby reduce the level of frataxin protein as related to the etiology of Friedreich's ataxia

Mapping Subunit Location on the Saccharomyces cerevisiae Origin Recognition Complex Free and Bound to DNA Using a Novel Nanoscale Biopointer

The Saccharomyces cerevisiae origin recognition complex (ORC) is composed of six subunits and is an essential component in the assembly of the replication apparatus. To probe the organization of this multiprotein complex by electron microscopy, each subunit was tagged on either its C or N terminus with biotin and assembled into a complex with the five other unmodified subunits. A nanoscale biopointer consisting of a short DNA duplex with streptavidin at one end was used to map the location of the N and C termini of each subunit. These observations were made using ORC free in solution and bound to the ARS1 origin of replication. This mapping confirms and extends previous studies mapping the sites of subunit interaction with origin DNA. In particular, we provide new information concerning the stoichiometry of the ORC-ARS1 complex and the changes in conformation that are associated with DNA binding by ORC. This versatile, new approach to mapping protein structure has potential for many applications

Architecture of the Bacteriophage T4 Replication Complex Revealed with Nanoscale Biopointers

Our previous electron microscopy of DNA replicated by the bacteriophage T4 proteins showed a single complex at the fork, thought to contain the leading and lagging strand proteins, as well as the protein-covered single-stranded DNA on the lagging strand folded into a compact structure. "Trombone" loops formed from nascent lagging strand fragments were present on a majority of the replicating molecules (Chastain, P., Makhov, A. M., Nossal, N. G., and Griffith, J. D. (2003) J. Biol. Chem. 278, 21276-21285). Here we probe the composition of this replication complex using nanoscale DNA biopointers to show the location of biotin-tagged replication proteins. We find that a large fraction of the molecules with a trombone loop had two pointers to polymerase, providing strong evidence that the leading and lagging strand polymerases are together in the replication complex. 6% of the molecules had two loops, and 31% of these had three pointers to biotin-tagged polymerase, suggesting that the two loops result from two fragments that are being extended simultaneously. Under fixation conditions that extend the lagging strand, occasional molecules show two nascent lagging strand fragments, each being elongated by a biotin-tagged polymerase. T4 41 helicase is present in the complex on a large fraction of actively replicating molecules but on a smaller fraction of molecules with a stalled polymerase. Unexpectedly, we found that 59 helicase-loading protein remains on the fork after loading the helicase and is present on molecules with extensive replication

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. 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 six hours 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

DNA replication in early S phase pauses near newly activated origins

During the S phase of the cell cycle, the entire genome is replicated. There is a high level of orderliness to this process through the temporally and topologically coordinated activation of many replication origins situated along chromosomes. We investigated the program of replication from origins initiating in early S phase by labeling synchronized normal human fibroblasts (NHF1) with nucleotide analogs for various pulse times and measuring labeled tracks in combed DNA fibers. Our analysis showed that replication forks progress 9–35 kilobases from newly initiated origins, followed by a pause in synthesis before replication resumes. Pausing was not observed near origins that initiated in the middle of S phase. No evidence for pausing near origins was found at the beginning of the S phase in glioblastoma T98G cells. Treatment with the S phase checkpoint inhibitor caffeine abrogated pausing in NHF1 cells in early S phase. This suggests that pausing may comprise a novel aspect of the intra-S phase checkpoint pathway or a related new early S checkpoint. Further, it is possible that the loss of this regulatory process in cancer cells such as T98G could be a contributing factor in the genetic instability that typifies cancers

Titanium dioxide nanoparticles activate the ATM-Chk2 DNA damage response in human dermal fibroblasts

The use of nanoparticles in consumer products increases their prevalence in the environment and the potential risk to human health. Although recent studies have shown in vivo and in vitro toxicity of titanium dioxide nanoparticles (nano-TiO2), a more detailed view of the underlying mechanisms of this response needs to be established. Here the effects of nano-TiO2 on the DNA damage response and DNA replication dynamics were investigated in human dermal fibroblasts. Specifically, the relationship between nano-TiO2 and the DNA damage response pathways regulated by ATM/Chk2 and ATR/Chk1 were examined. The results show increased phosphorylation of H2AX, ATM, and Chk2 after exposure. In addition, nano-TiO2 inhibited the overall rate of DNA synthesis and frequency of replicon initiation events in DNA combed fibers. Taken together, these results demonstrate that exposure to nano-TiO2 activates the ATM/Chk2 DNA damage response pathway

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 to 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 and 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

Human CST promotes telomere duplex replication and general replication restart after fork stalling

Mammalian CST (CTC1-STN1-TEN1) associates with telomeres and depletion of CTC1 or STN1 causes telomere defects. However, the function of mammalian CST remains poorly understood. We show here that depletion of CST subunits leads to both telomeric and non-telomeric phenotypes associated with DNA replication defects. Stable knockdown of CTC1 or STN1 increases the incidence of anaphase bridges and multi-telomeric signals, indicating genomic and telomeric instability. STN1 knockdown also delays replication through the telomere indicating a role in replication fork passage through this natural barrier. Furthermore, we find that STN1 plays a novel role in genome-wide replication restart after hydroxyurea (HU)-induced replication fork stalling. STN1 depletion leads to reduced EdU incorporation after HU release. However, most forks rapidly resume replication, indicating replisome integrity is largely intact and STN1 depletion has little effect on fork restart. Instead, STN1 depletion leads to a decrease in new origin firing. Our findings suggest that CST rescues stalled replication forks during conditions of replication stress, such as those found at natural replication barriers, likely by facilitating dormant origin firing

Accumulation of true single strand breaks and AP sites in base excision repair deficient cells

Single strand breaks (SSBs) are one of the most frequent DNA lesions caused by endogenous and exogenous agents. The most utilized alkaline-based assays for SSB detection frequently give false positive results due to the presence of alkali-labile sites that are converted to SSBs. Methoxyamine, an acidic O-hydroxylamine, has been utilized to measure DNA damage in cells. However, the neutralization of methoxyamine is required prior to usage. Here we developed a convenient, specific SSB assay using alkaline gel electrophoresis (AGE) coupled with a neutral O-hydroxylamine, O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (OTX). OTX stabilizes abasic sites (AP sites) to prevent their alkaline incision while still allowing for strong alkaline DNA denaturation. DNA from DT40 and isogenic polymerase β null cells exposed to methyl methanesulfonate were applied to the OTX-coupled AGE (OTX-AGE) assay. Time-dependent increases in SSBs were detected in each cell line with more extensive SSB formation in the null cells. These findings were supported by an assay that indirectly detects SSBs through measuring NAD(P)H depletion. An ARP-slot blot assay demonstrated a significant time-dependent increase in AP sites in both cell lines by 1 mM MMS compared to control. Furthermore, the Pol β-null cells displayed greater AP site formation than the parental DT40 cells. OTX use represents a facile approach for assessing SSB formation, whose benefits can also be applied to other established SSB assays