Studies on the repair and conformation of DNA containing O6-alkylguanine and O4-alkylthymine

The carcinogenic N-nitroso compounds alkylate DNA. Among the different products of alkylation, O6-alkylguanine and O4-alkylthymine have attracted most of the attention since they are highly mutagenic and a correlation exists between their formation and persistence, and oncogenesis in animal model systems. Both DNA adducts in E. coli and at least O6-alkylguanine in mammalian cells are repaired by enzymes, the so-called O6-alkylguanine-DNA-alkyltransferases. Based on the HPLC separation of short, self-complementary oligonucleotides containing O6-methylguanine, O6-ethylguanine, or O4-methylthymine from the respective parent non-alkylated oligomers, the rate constants for their repair by the E. coli ada and ogt and the human alkyltransferases were determined. Although all alkyltransferases were able to repair O6-methylguanine, O6-ethylguanine and O4-methylthymine, the relative efficiencies were found to differ significantly. Using an immunoprecipitation assay, the rates of repair of an O6-methylguanine residue in various positions in 15 base-pair DNA duplexes were measured. The sequence of the oligomers was that of the rat H-ras sequence around codon 12 and the rates of repair were found to vary up to 25-fold depending on the sequence flanking the methylguanine. An O6-methylguanine in the second position of the GGA codon 12 was the least well repaired. The combination of this slow repair and sequence selectivity in alkylation appears to be the explanation of the selective mutation of this position observed in rat mammary tumours. The avidity constants between antibody and O6-methylguanine were also dependent on the sequence flanking the adduct, with the most rapidly repaired being those most easily bound to the antibody. It is suggested that the rate of repair is a reflection of the conformation of the oligomers containing O6-methylguanine. An unusual feature of DNA which is often associated with protein-DNA interactions is DNA curvature. A characteristic of curved DNA is that it has less electrophoretic mobility than normal DNA. In order to assess if alkylated adducts in DNA induce DNA curvature or flexibility, DNA duplexes containing O4-alkylthymine or O6-methylguanine were synthesized and self-ligated to form multimers with the alkylated bases out of phase (16 base-pairs apart) or in phase (21 base-pairs apart) with the helical repeat of DNA. All the sequences containing O4-alklylthymine migrated more slowly than expected in a non-denaturing polyacrylamide gel. In general the effect was seen when the alkylated base was out of phase or in phase with the helical repeat suggesting that the altered base-pair confers flexibility which is largely isotropic, i.e has no preferred direction, rather than anisotropic flexibility or bending. The effect of O4-methylthymine in the mobility of the oligonucleotides was much greater than that of O6-methylguanine and the effect of O4-ethylthymine slightly greater than that of O4-methylthymine. DNA duplexes containing O4-alkylT:A base-pairs were more retarded, and had lower thermal point (Tm) than DNA duplexes containing O4-alkylT:G base-pairs

The Mechanisms of DNA Replication

DNA replication is a fundamental part of the life cycle of all organisms. Not surprisingly many aspects of this process display profound conservation across organisms in all domains of life. The chapters in this volume outline and review the current state of knowledge on several key aspects of the DNA replication process. This is a critical process in both normal growth and development and in relation to a broad variety of pathological conditions including cancer. The reader will be provided with new insights into the initiation, regulation, and progression of DNA replication as well as a collection of thought provoking questions and summaries to direct future investigations

DNA damage tolerance in mammalian cells

DNA is susceptible to both exogenous and endogenous damaging agents. Damage is constantly reversed by a wide range of DNA repair pathways. Lesions which escape such repair may cause nucleotide mis-pairing and stalled replication, resulting in mutagenesis and cell death, respectively if left unresolved. Stalled replication is particularly dangerous because replication fork collapse can lead to double-strand breaks (DSBs) and chromosome rearrangement, a hallmark of cancer. DNA damage tolerance (DDT) is defined as a mechanism that allows DNA synthesis to occur in the presence of replication-blocking lesions. DDT, also known as post-replication repair (PRR) in yeast, has been well characterized in the lower eukaryotic model Saccharomyces cerevisiae to consist of error-free and error-prone (mutagenic) pathways. Mono-ubiquitination of proliferating cell nuclear antigen (PCNA) by the Rad6-Rad18 complex promotes mutagenesis by recruiting low fidelity translesion synthesis (TLS) polymerases, while continual Lys63-linked poly-ubiquitination of PCNA by the Mms2-Ubc13-Rad5 complex promotes error-free lesion bypass. Since most of the genes involved in DNA metabolism are conserved within eukaryotes, from yeast to human, I tested the hypothesis that mammalian cells also possess two-pathway DDT in response to DNA damage. Namely, the error-free pathway is dependent on the Ubc13-Mms2 complex, while the error-prone pathway utilizes the TLS polymerases, such as Rev3. By utilizing cultured mammalain cells and producing antibodies against human Ubc13, Mms2 and Rev3, I was able to show that all three proteins associate with PCNA in S-phase cells, and that this association is enhanced following DNA damage. Ubc13-Mms2 association with PCNA was enhanced in response to DSBs. Furthermore, suppression of Ubc13 or Mms2 using interfering RNA technology resulted in increased spontaneous DSBs. In response to UV exposure, Rev3 co-localized with PCNA and two other TLS polymerases, Rev1 and Pol-ƒØ, at the damage site. UV-induced Rev3 nuclear focus formation was dependent on Rev1 but independent of Pol-£b. Surprisingly, over-expression of Pol-£b was sufficient to induce spontaneous Rev3 nuclear foci. It was further demonstrated that Rev1 and Pol-ƒØ were independently recruited to the damage site and did not require Rev3. These observations support and extend the polymerase switch model which regulates the activity of the replicative and TLS polymerases. Finally, simultaneous suppression of Rev3 along with Ubc13 or Mms2 resulted in a synergistic sensitivity to UV, whereas simultaneous suppression of Ubc13 and Pol-ƒØ resulted in an additive effect. These results are consistent with those in yeast cells, implying a comparable mammalian two-pathway DDT model. Additional interesting observations were made. Firstly, Ubc13 interacts with Uev1A, a close homolog of Mms2, which is involved in the NF-£eB signaling pathway independent of DNA damage. Secondly, Rev3 appears to be excluded from the nucleus in a fraction of low passage normal non-S-phase cells, whereas in tumor derived cell lines, Rev3 is consistently enriched in the nucleus independent of cell cycle stage. Finally, Rev3 is elevated during mitosis and associates with condensed chromosomes, suggesting a possible novel role in mitosis. Consistent with this notion, chronic ablation of Rev3 resulted in cell death with inappropriate chromosome segregations. The above preliminary observations require further investigation

Tyrosine Phosphorylation of p68 RNA Helicase Promotes Metastasis in Colon Cancer Progression

The initiation of cancer metastasis usually requires Epithelial-Mesenchymal Transition (EMT), by which tumor cells lose cell-cell interactions and gain the ability of migration and invasion. Previous study demonstrated that p68 RNA helicase, a prototypical member of the DEAD-box RNA helicases, functions as a mediator to promote platelet-derived growth factor (PDGF)-induced EMT through facilitating nuclear translocation of β-catenin in colon cancer cells. In this context, p68 RNA helicase was found to be phosphorylated at the tyrosine 593 residue (referred as phosphor-p68) by c-Abl kinase, and this phosphorylation is required for the activation of β-catenin signaling and the consequent EMT. The phosphor-p68 RNA helicase-mediated EMT was characterized by the repression of an epithelial marker, E-cadherin, and the upregulation of a mesenchymal marker, Vimentin. E-cadherin, a major cell-cell adhesion molecule that is involved in the formation of adherens junctions, has been shown to sequester β-catenin at the cell membrane and thus inhibit its transcriptional activity. The functional loss of E-cadherin is the fundamental event of EMT. Despite the role of phosphor-p68 RNA helicase in regulating nuclear translocation of β-catenin, whether phosphor-p68 is involved in the regulation of E-cadherin remains unknown. Here, our data indicated that phosphor-p68 RNA helicase initiated EMT by transcriptional upregulation of Snail1, a master transcriptional repressor of E-cadherin. The data suggest that phosphor-p68 RNA helicase displaced HDAC1 from the chromatin remodeling MBD3:Mi-2/NuRD complex at the Snail1 promoter, thereby activating the transcription of Snail1. In the xenograft tumor model, abolishing the phosphorylation of p68 RNA helicase by the expression of Y593F mutant resulted in a significant reduction of metastatic potential in human colon cancer cells. Analyses in the colon cancer tissues also revealed that the tyrosine 593 phosphorylation level of p68 RNA helicase is substantially enhanced in the tumor tissues comparing to that in the corresponding normal counterparts, suggesting a correlation of phosphor-p68 and tumor progression. In conclusion, we showed that tyrosine phosphorylation of p68 RNA helicase positively correlated to the malignant status of colon cancer progression. The molecular basis behind this correlation could be partly through the transcriptional regulation of Snail1

Role of replicative primase in lesion bypass during DNA replication

Maintenance of genome integrity and stability is fundamental for any form of life. This is complicated as DNA is highly reactive and always under attack from a wide range of endogenous and exogenous sources which can lead to different damages in the DNA. To preserve the integrity of DNA replication, cells hav evolved a variety of DNA repair pathways. DNA damage tolerance mechanisms serve as the last line of defence to rescue the stalled replications forks. TLS, error-prone type of DNA damage tolerance, acts to bypass DNA lesions and allows continuation of DNA replication. Surprisingly majority of archaeal species lack canonical TLS polymerases. This poses a question as to how archaea restart stalled replication in the absence of TLS or lesion repair pathways. This thesis establishes that archaeal replicative primase (PriS/L), a member of the archaeo-eukaryotic primase (AEP) superfamily, possessing both primase and polymerase activities, is able to bypass the most common oxidative damages and highly distorting lesions caused by UV radiation. It has been postulated that archaeal replicative polymerases (Pol B and Pol D family Pols) can bind tightly to the deaminated bases uracil and cause replication fork stalling four bases prior to dU. A specific mechanism for resuming replication of uracil containing DNA by PriS/L is suggested in this thesis. In this thesis, we also reported how the enzymatic activities of archaeal PriS/L are regulated. Here, it is demonstrated that in contrast to archaeal replicative polymerases, single-strand binding proteins (RPA) significantly limit the polymerase activity of PriS/L. The remaining results chapter interrogates the possible interactions between PriS/L and RPA. Finally, the attempts to reconstitute an archaeal CMG complex in vitro, with the aim of shedding light on the role of archaeal replicative primase in replication-specific TLS are described

Evaluation of the DNA replication licensing machinery as an anti-proliferative target.

Cancer is the second leading cause of death in the developed world. However despite significant advances in chemotherapy during the latter half of the 20th Century, the impact on mortality has been modest. There is therefore a profound need for the identification of novel strategies to treat cancer. The DNA replication licensing pathway, which co-ordinates the decision to initiate DNA replication, has recently emerged as a potential anti-cancer target. The early dysregulation of the replication licensing machinery during tumourigenesis suggests that agents targeting this pathway may have high efficacy in tumour cells. However, the response of normal cells to such agents must also be considered. Here I show that withdrawal of cells from the mitotic cell cycle into the out-of-cycle states of quiescence and differentiation is tightly coupled to down-regulation of the DNA replication licensing machinery. Importantly, stem/progenitor cells of self- renewing tissues display an unlicensed replication phenotype. These results indicate that normal out-of-cycle functional, differentiated and stem/progenitor cell populations will be refractory to agents targeting the replication licensing machinery. Rapidly proliferating normal cell populations can suffer severe genotoxic and cytotoxic damage in response to chemotherapy. Here I show that inhibition of origin licensing in normal proliferating cells invokes the reversible activation of a putative origin licensing checkpoint which stalls cells in Gl until the block to origin licensing is removed, thereby protecting cells from damage. In contrast, transformed cells respond to inhibition of origin licensing by inducing apoptosis, suggesting that origin licensing inhibitors may represent highly specific cancer killing agents. Finally, I have utilised a cell-free DNA replication and chromatin-binding assay to analyse the biochemical properties of two potential lead compounds: the endogenous origin licensing repressor geminin and a viral pathogen HPV1 E4. Collectively, these studies reinforce the concept that the inhibition of DNA replication licensing represents a novel chemotherapeutic strategy to combat cancer

The role of SMARCAD1 during replication stress

Heterozygous mutations in BRCA1 or BRCA2 predispose carriers to an increased risk for breast or ovarian cancer. Both BRCA1 and BRCA2 (BRCA1/2) play an integral role in promoting genomic stability through their respective actions during homologous recombination (HR) mediated repair and stalled replication fork protection from nucleolytic degradation. SMARCAD1 (SD1) is a SWI/SNF chromatin remodeler that has been implicated in promoting long-range end resection and contributes to HR. Using human cell lines, we show that SMARCAD1 promotes nucleolytic degradation in BRCA1/2-deficient cells dependent on its chromatin remodeling activity. Moreover, SMARCAD1 prevents DNA break formation and promotes fork restart at stalled replication forks. These studies identify a new role for SMARCAD1 at the replication fork. In addition to the work presented here, I discuss a method for introducing stop codons (nonsense mutations) into genes using CRISPR-mediated base editing, called iSTOP, and provide an online resource for accessing the sequence of iSTOP sgRNASs (sgSTOPs) for five base editor variants (VQR-BE3, EQR-BE3, VRER-BE3, SaBE3, and SaKKH-BE3) in humans and over 3 million targetable gene coordinates for eight eukaryotic species. Ultimately, with improvements to CRISPR base editors this method can help model and study nonsense mutations in human disease