No description available.

No description available.

Radiotherapy continues to be a primary modality in the treatment of cancer. DNA damage induced by radiation can promote apoptosis as well as both autophagy and senescence, where autophagy and senescence can theoretically function to prolong tumor survival. A primary aim of this work was to investigate the hypothesis that autophagy and/or senescence could be permissive for DNA repair, thereby facilitating tumor cell recovery from radiation-induced growth arrest and/or cell death. In addition, studies were designed to elucidate the involvement of autophagy and senescence in radiation sensitization by PARP inhibitors and the re-emergence of a proliferating tumor cell population. In the context of this work, the relationship between radiation-induced autophagy and senescence was also determined. Studies were performed using DNA repair proficient HCT116 colon carcinoma cells and a repair deficient Ligase IV (-/-) isogenic cell line. Irradiation promoted a parallel induction of autophagy and senescence that was strongly correlated with the extent of persistent H2AX phosphorylation in both cell lines; however inhibition of autophagy failed to suppress senescence, indicating that the two responses were dissociable. Irradiation resulted in a transient arrest in the HCT116 cells while arrest was prolonged in the Ligase IV (-/-) cells; however, both cell lines ultimately recovered proliferative function, which may reflect maintenance of DNA repair capacity. The PARP inhibitors (Olaparib) and (Niraparib) increased the extent of persistent DNA damage induced by radiation as well as the extent of both autophagy and senescence; neither cell line underwent significant apoptosis by radiation alone or in the presence of the PARP inhibitors. Inhibition of autophagy failed to attenuate radiation sensitization, indicating that autophagy was not involved in the action of the PARP inhibitors. As with radiation alone, despite sensitization by PARP inhibition, proliferative recovery was evident within a period of 10-20 days. While inhibition of DNA repair via PARP inhibition may initially sensitize tumor cells to radiation via the promotion of senescence, this strategy does not appear to interfere with proliferative recovery, which could ultimately contribute to disease recurrence.

Proliferating cell nuclear antigen (PCNA) is a sliding clamp that acts as a central co-ordinator for mismatch repair as well as DNA replication. Loss of Elg1, the major subunit of the PCNA unloader complex, causes over-accumulation of PCNA on DNA and also increases mutation rate, but it has been unclear if the two effects are linked. In this study, I showed that timely control of PCNA retention on DNA by Elg1 replication factor C-like complex (Elg1-RLC) ensures correct mismatch repair. Although premature unloading of PCNA generally increases mutation rate, PCNA mutants PCNA-R14E and PCNA-D150E that spontaneously fall off DNA attenuate the mutator phenotype of elg1Δ. In contrast, PCNA-D21K that accumulates on DNA due to enhanced electrostatic PCNA-DNA interactions exacerbates the elg1Δ mutator phenotype. Next, I addressed how accumulation of PCNA on DNA increases mutation rate. Epistasis analysis suggests that PCNA over-accumulation on DNA predominantly prevents the Msh2-Msh6-dependent and Exo1-independent mismatch repair pathways. In elg1Δ, over-retained PCNA hyper-recruits the Msh2-Msh6 mismatch recognition complex through its PCNA-interacting peptide motif, causing accumulation of mismatch repair intermediates. The results suggest that PCNA retention controlled by the Elg1-RLC is critical for efficient mismatch repair: PCNA needs to be on DNA long enough to enable mismatch repair, but if it is retained too long it interferes with downstream repair steps.

Melanoma is one of the most common cancers in the world. For primary melanoma, early diagnosis and surgical excision are effective treatments but, despite the new targeted therapies and immunotherapies, there is still a need for more effective treatment options to improve overall survival for patients with metastatic melanoma. Chemotherapy with genotoxic agents remains the main approach for most cancers, but DNA repair pathways in cancer cells reduce their effectiveness. So disruption of key DNA repair pathways, such as nucleotide excision repair (NER), could be an effective option to combine with chemotherapy for melanoma. The structure-specific endonuclease ERCC1-XPF, which heterodimerises through the C-terminal helix-hairpin-helix (HhH)2 domains of both proteins, is essential for NER. The aim of my project was to determine the mechanism involved in regulating the level of the ERCC1-XPF heterodimer with a view to disrupting NER activity. The project started by determining the ERCC1 and XPF response in six melanoma cell lines to the chemotherapeutic cisplatin at the mRNA and protein levels. Although the mRNA and protein levels of both ERCC1 and XPF increased, there was variable consistency between the cell lines, raising the possibility that post translational modification may play an important role in the regulation of ERCC1- XPF activity. We chose to focus on ubiquitination, because it can affect a protein’s activity at both expression and activation levels and several examples of ubiquitinated DNA repair proteins were known. In the pilot study we found that ERCC1 was accumulated after proteasome inhibitor treatment and decreased by treatment with a translation inhibitor in two melanoma cell lines, suggesting that ERCC1 may be ubiquitinated. By cotransfecting His-tagged ubiquitin and non-tagged ERCC1 constructs into melanoma cells and performing an ubiquitin assay, we found that ERCC1 was degraded by the proteasome system through polyubiquitination or multiple monoubiquitination. To determine the nature of the ubiquitination type, we mutated each of the seven Lys residues on ubiquitin and carried out additional assays with ubiquitin single and combination mutants, and discovered that Lys33 was most likely involved in the proteasome dependent degradation of ERCC1. By immunoprecipitation with an antibody to linear ubiquitin from melanoma cell extracts containing a ubiquitin construct with all seven Lys residues mutated to Arg, we found that the N-Met of ubiquitin was also most likely involved in ERCC1 ubiquitination. To determine which Lys of ERCC1 is used by ubiquitin, we did another series of in vivo ubiquitin assays with full length and truncated ERCC1 constructs and found that the key amino acid is most likely within the C-terminal XPF binding domain of ERCC1. By cotransfecting the full length ERCC1 and ERCC1 truncation constructs together with full length XPF, we showed that the ubiquitination of ERCC1 was not an artefact resulting from overexpression of ERCC1 alone and that the stability of XPF was dependent on the overexpression and stability of ERCC1. We then made single lysine and lysine combination mutants in the XPF binding domain of ERCC1 and found that none of the lysines were essential for ubiquitination of ERCC1, indicating that a non- lysine amino acid might be used for ubiquitination. However, using a transfection-based NER assay in ERCC1-deficient cells, we found that ubiquitination of Lys 295 could be involved in regulating the DNA repair activity of ERCC1-XPF. The in vivo ubiquitin assay result after cotransfection of ERCC1 and XPF, which showed that XPF was dependent on the presence of ERCC1 for stability, but not vice versa, was inconsistent with previous published data suggesting that heterodimerization was essential for the stability of both proteins. Instead we hypothesised that homodimerization of ERCC1 might be another mechanism to keep ERCC1 stable and obtained evidence for this at the overexpression level by immunoprecipitation following cotransfection of Myc-tagged ERCC1 and Flag-tagged ERCC1 or ERCC1 truncations, which was supported at the endogenous expression level by size exclusion chromatography on melanoma cell extracts to identify ERCC1 in different molecular weight fractions. In the previous in vivo ubiquitin assay, we found that levels of transfected full length ERCC1 and XPF were dramatically reduced by cotransfection with the Flag-tagged ERCC1 (220-297) construct that just contains the XPF binding domain of ERCC1. This led to another hypothesis, that the ERCC1 (220-297) peptide can decrease endogenous levels of ERCC1 and XPF and so be a potential drug in combination with cisplatin chemotherapy. This hypothesis was verified in stable transgenic cell lines expressing ERCC1 (220-297) which showed reduced levels of ERCC1 and XPF and of NER and increased sensitivity to cisplatin and UV irradiation. Based on the above results and supporting bioinformatics analysis we have made the following conclusions: ERCC1 is regulated by the ubiquitin-proteasome degradation pathway through linkages most likely involving Lys33 and N-Met; the XPF binding domain is most likely the key domain for ERCC1 ubiquitination; XPF stability is dependent on the presence of ERCC1 and seems affected by ERCC1 ubiquitination; ERCC1 seems to be ubiquitinated in a non-conventional lysine-independent manner and ubiquitination of Lys 295 might be involved in the regulation of the DNA repair activity of ERCC1- XPF; homodimerization is most likely a novel mechanism to keep ERCC1 stable; the ERCC1 (220-297) peptide can destabilise both ERCC1 and XPF and could be a potential drug in combination with genotoxic therapies.

This study was conducted at the University of Massachusetts Medical Center in the Volkert laboratory. Human alkyl adenine DNA glycosylase (hAAG) is a DNA repair enzyme that repairs alkylated DNA bases. hAAG was cloned in 1991 and a second isoform was classified in 1994. The difference between the two isoforms of hAAG is an alternate spliced first exon. Both isoforms of the hAAG gene were present in the Volkert laboratory collection, however the second isoform (hAAG-2) was phenotypically different than the first and became the first focus of this study. Using the improperly functioning isoform as a template, and constructing a 5' primer with the identical upstream sequence as the functioning isoform (hAAG-1), a phenotypically similar gene was constructed by PCR. The new isoform (hAAG-2) was cloned into an expression vector and its activity as a DNA repair agent was studied. A second version of hAAG-2 was also constructed, incorporating a histidine tag for protein purification and identification purposes. Efforts included using the ability of hAAG to complement glycosylase deficient alkA tagA E. coli double mutant strains to assess and to compare the ability of the two isoforms of hAAG and to determine if the histidine tag affected function. The ability of hAAG to rescue cells from exposure to a variety of DNA damaging agents was studied by inducing each isoform and analyzing the sensitivity of the cells to increased doses of DNA damaging agents. Both hAAG-1 and hAAG-2 were able to restore the wild type resistance of the alkA and tag genes when exposed to the alkylating agents MNNG and MMS. In order to study the ability of hAAG to repair alkyl lesions larger than methyl groups, it was necessary to inactivate the uvrA dependent nucleotide excision repair gene. In E. coli, methyl lesions are repaired primarily by glycosylases, while nucleotide excision repairs bulky lesions. Thus, in order to detect hAAG activity on these types of damage, it was necessary to inactivate the bacterial uvrA gene. Each isoform of hAAG was transformed into a triple mutant strain deficient in alkA tagA and uvrA, then exposed to CNU, BCNU, and Mitomycin C. Each of these DNA damaging agent caused increased toxicity in the presence of hAAG. hAAG-1 expressed in the alkA tag double mutant strain was exposed to Mitomycin C and showed greater resistance than hAAG-1 expressed in the alkA tag uvrA triple mutant. In fact, in the nucleotide excision proficient strain, expression increased Mitomycin C resistance above that seen in the control, suggesting that glycosylase activity may function in a partnership with nucleotide excision repair and that the two isoforms of hAAG have subtle differences. An ompT protease knockout host strain was constructed using P1-transduction and used to examine protein products. hAAG-2 was inserted into the pBlueScript plasmid so that the gene could be regulated by the T7 promoter for use beyond the scope of this thesis. A protein synthesis time course assay was conducted to determine the expression levels of hAAG-1 and hAAG-2 when induced by IPTG. Immunoblot detection of the histidine tag was used to measure expression levels of each isoform.

DNA interstrand cross-links are a form of genomic damage that cause a block to replication and transcription of DNA in cells and cause lethality if unrepaired. Chemical agents that induce cross-links are particularly effective at inactivating rapidly dividing cells and, because of this, have been used to treat hyperproliferative skin disorders such as psoriasis as well as a variety of cancers. However, evidence for the removal of cross-links from DNA as well as resistance to cross-link-based chemotherapy suggests the existence of a cellular repair mechanism. Characterizing the pathways involved in DNA interstrand cross-link repair has been challenging due to the inherent structure of the damage as it precludes the use of an undamaged, complementary strand of DNA as a template for repair. A number of models of cross-link repair have been proposed based on the identification of hypersensitive repair mutants as well as biochemical evidence that specific repair enzymes are capable of incising cross-linked structures from DNA. Together, these models have suggested the involvement of multiple repair pathways--such as nucleotide exicision repair, translesion synthesis, recombination of double-strand breaks, and base excision repair--operating in sequential steps to correct the damage. Most of the studies from which these models arose are complicated by the fact that cross-linking agents induce multiple forms of damage or they lack in vivo confirmation of how the repair phenomenon occurs in organisms. In this study, I use Escherichia coli as a model organism to examine the involvement of the aforementioned pathways in DNA interstrand cross-link repair in vivo. This organism was useful in early cross-link studies and, with its highly conserved repair processes, maintains the potential for delineating how cross-links are removed in higher organisms. In Chapter I, I introduce background information on different cross-linking agents, the complications of studying cross-link repair, and the candidate repair pathways that have been implicated to date. In Chapter II I demonstrate that there is a limited involvement of the nucleotide excision repair helicase, translesion polymerases, and double-strand break repair enzymes through survival analysis of cells defective in these proteins. For this analysis, I use 8-methoxypsoralen plus UVA as a cross-linking agent and angelicin plus UVA as a monofunctional comparator. The observation that uvrD mutants-- defective in helicase II of nucleotide excision repair--were nearly as resistant to 8-methoxypsoralen-induced damage as wild type cells led me to examine the incision rate of cross-links from endogenous plasmid DNA. Surprisingly, cross-links were not efficiently removed from DNA in uvrD mutants relative to wild type cells. These seemingly contradictory results were rectified when I quantified cross-link formation in cell cultures and revealed that as few as one cross-link per chromosome can inactive wild type cells, a lethal quantity that is lower than what has been previously reported. Taken together, these observations suggest that although cross-links are incised in wild type cells, repair is still not a highly productive event in E. coli. In Chapter III I examine the involvement of the base excision repair pathway in cross-link repair and demonstrate that Nth and Fpg Glycosylases, Xth and Nfo AP-Endonucleases sensitize Escherichia coli to psoralen-induced DNA damage. This is shown by comparative survival analysis in angelicin plus UVA and 8-methoxypsoralen plus UVA treatment whereby nth-, fpg-, and xth-mutants are each more resistant than wild type cells to either treatment. This suggests that when these gene products are present they impact the production or removal of monoadducts. nfo-mutants were different in that the cells were only hyperresistant to 8-methoxypsoralen monoadducts and cross-links, either implying that the Nfo enzyme interacts specifically with psoralen monoadducts rather than angelicin monoadducts or that the enzyme impedes cross-link removal. Finally, in Chapter IV a summary of the results is provided as well as future directions that may be explored following this study.

BRCA1 is a caretaker of genome integrity with various molecular functions, which are required for development and tumor suppression. These include the homology-directed repair (HDR) of DNA double strand breaks, stalled replication fork protection (SFP), transcription, chromatin remodeling and cell cycle checkpoint control. Recent studies reported that BRCA1 is required for reprogramming to pluripotency, but its specific role remains unknown. In this work, we use separation of function mutants for the roles of BRCA1 in HDR and SFP to show that BRCA1 is required to repair replication-associated DNA double strand breaks by homologous recombination during reprogramming. Deficiency in SFP proved inconsequential to induced pluripotent stem (iPS) cell generation and cells with this phenotype did not experience reduced reprogramming. Thus, the primary limiting factor for the transition to pluripotency is a specific class of DNA damage: double strand breaks, likely occurring in late replicating regions which require repair by homologous recombination. These findings identify an important role of DNA damage, linked to the progression of DNA replication, in limiting cell type transitions during reprogramming. Most studies on iPS cell generation have focused on gene expression as a limiting step, in part due to the wide availability of tools to analyze transcription. Since the progression of DNA replication and DNA damage during S-phase are cell type specific, we have started the development of a sequencing platform to map various aspects of replication progression, such as origin usage, polymerase direction,pausing and stalling. In this work, we demonstrate that nucleotide analogs, incorporated during DNA synthesis in mammalian cells, can be detected by Nanopore sequencing.

No description available.

Escherichia coli double-strand uracil-DNA glycosylase (Dug) was purified to apparent homogeneity from bacteria that were defective in uracil-DNA glycosylase (Ung). After cloning the dug gene, recombinant Dug was overexpressed, purified, and characterized with respect to activity, substrate specificity, product DNA binding, and mechanism of action. Purified Dug excised both uracil and ethenocytosine specifically from double-stranded DNA substrates. One distinctive characteristic of Dug was that the purified enzyme removed a near stoichiometric amount of uracil from DNA containing U/G mispairs. The observed lack of turnover was attributed to tight binding of Dug to the apyrimidinic-site (AP) contained in the DNA reaction product. Catalytic activity was stimulated in the presence of E. coli endonuclease IV that caused AP-site incision and dissociation of Dug. By using enzyme complementation experiments, Dug was shown to initiate uracil-initiated base excision repair (BER) in E. coli (ung) cell-free extracts. The relative rate of repair of uracil- and ethenocytosine-containing DNA in isogenic E. coli cells that were proficient or deficient in Ung and/or Dug was measured using a novel competition assay. Complete ethenocytosine-initiated BER displayed an absolute requirement for Dug and occurred at the same rate as uracil-initiated BER in the presence of both Ung and Dug. However, the rate of Dug-mediated ethenocytosine-DNA repair was 8-fold faster than that of uracil-DNA mediated by Dug. The distribution of BER patch sizes associated with both uracil- and ethenocytosine-containing DNA showed similar results. In both cases, DNA repair synthesis utilized predominantly a long patch BER mechanism involving the incorporation of 2-20 nucleotides. A previously unidentified "very long patch" mechanism of BER involving the incorporation of more than 200 nucleotides was identified and shown to be mediated by DNA polymerase I. The rate-limiting step associated with uracil-initiated BER was found to involve DNA ligase and the distribution of BER patch size was modulated by the ratio of DNA polymerase I and DNA ligase. The fidelity of DNA repair synthesis associated with complete uracil-DNA BER was measured using E. coli cell-free extracts that were proficient or deficient in Ung activity and determined to be 5.5 x 10������ and 19.7 x 10������, respectively. / Graduation date: 2003