Browsing by Subject "DNA replication"

Now showing 1 - 5 of 5
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    Chemistry and Biology of DNA-protein cross-links
    (2019-02) Ji, Shaofei
    DNA-protein cross-links (DPCs) are ubiquitous DNA lesions formed when proteins become covalently trapped on DNA strands upon exposure to various endogenous, environmental and chemotherapeutic agents. Because of their considerable size, DPCs interfere with the progression of replication and transcription machineries, potentially contributing to mutagenesis and carcinogenesis. However, unlike small DNA lesions of which the biological consequences and repair mechanisms have been well characterized, biological effects and repair mechanism of DPC lesions remain to be established. A significant challenge in the field is the structural diversity of DPC lesions and the scarcity of experimental mythologies to create site-specific DNA-protein conjugates. The main objective of this thesis was to synthesize model DPC and to investigate their biological consequences and repair mechanisms. In Chapter II, we discovered, characterized and quantified 5-formylcytosine(5fC) mediated DNA-histone conjugates in human cells. 5-Formylcytosine (5fC) is an endogenous DNA modification enzymatically generated in the genome as an oxidation product of 5-methyl-dC (5mC). While 5mC is known to be an epigenetic mark that controls the levels of gene expression, the biological functions of 5fC are incompletely understood. In this chapter, we discovered that 5fC bases in DNA readily form Schiff base conjugates with Lys side chains of nuclear proteins such as histones, forming covalent DNA-protein conjugates. Isotope dilution nanoLC-ESI-MS/MS methodology was employed to detect and quantify 5fC-lys conjugate in human cells. We hypothesize that reversible 5fC-histone cross-linking contributes to epigenetic signaling, transcriptional regulations and chromatin remodeling. After the discovery of 5fC-mediated DNA-histone crosslinks in mammalian cells, we investigated their effects on DNA replication in Chapter III. DNA substrates containing site-specific DPCs were subjected to in vitro translesion synthesis (TLS) in the presence of TLS DNA polymerases. We found that DPCs containing various full-length proteins conjugated to DNA via the C-5 position of cytosine completely blocked human DNA polymerases, while the corresponding lesions containing shorter peptides were bypassed by translesion synthesis (TLS) polymerases. These results are consistent with the proposed DPC repair pathway in the literature, in which full-length DPCs are subjected to proteolytic degradation to generate short DNA-peptide cross-links, which may serve as substrates for translesion synthesis. In addition, our steady-state kinetics analysis and mass- spectrometry-based sequencing and quantification revealed that the bypass of DNA- peptide cross-links by human TLS polymerases was highly error-prone, introducing significant amounts of C to T and deletion mutations. In Chapter IV, we investigated the effects of DPCs on transcription using two model DPCs where the proteins are conjugated to the C5 position of cytosine or the C7 position of 7-deazaguanine. The latter serves as a hydrolytically stable model of the N7- guanine lesions, which commonly form upon exposure to bis-electrophiles such antitumor agents. We found that full-length proteins cross-linked to either 5fC or 7-deazaguanine completely blocked T7 RNA polymerase, while relatively short peptide cross-links were bypassed, although with low efficiency. Interestingly, the two model DPCs exhibited completely different mutagenic patterns are revealed by PCR and mass spectrometry based assays. While the bypass of peptide cross-linked to 7-deaza-G by T7 RNA polymerases induced very small numbers of mutations, transcription past peptide lesions conjugated to C-5 of C induced significant amounts of C to T transcriptional mutations. In Chapter V, we investigated the effects of 5fC-mediated DNA-peptide/protein cross-links on transcription and its potential repair by nucleotide excision repair (NER) in living cells. To accomplish this goal, structurally defined DPCs were site-specifically incorporated into plasmid molecules, which was then transfected into wild type cells or cells deficient in NER. RT-PCR and LC-MS/MS based strategy was then employed to quantitatively study the effects of DPC lesions on efficiency and fidelity of transcription in mammalian cells. We found that the presence of peptide cross-links conjugated to C-5 of cytosine significantly inhibited DNA transcription in human embryonic kidney cells. However, in contrast to our in vitro results, no transcriptional mutagenesis was observed. In addition, we compared the transcription bypass efficiencies of DpC lesions in wild-type and NER-deficient cell-lines, and also conducted the in vitro NER assays using cell-free extracts from human HeLa cells. Collectively, our data suggested that 5fC-mediated DNA- peptide cross-links are poor NER substrates, requiring a different pathway for their repair. Recent studies suggested that the bulky DPCs in cells are proteolytically processed to shorter DNA-peptide cross-links before they can be tolerated by translation synthesis mechanism or removed by nucleotide excision repair. DPCs can block DNA replication, signaling for recruitment of specialized metalloprotease (Spartan). However, the mechanisms of protease-mediated DPC digestion in the absence of DNA replication are incompletely understood. In Chapter VI, we employed an immunoprecipitation(IP)-PCR methodology to demonstrate that DPCs present on non-replicating plasmids are rapidly ubiquitinylated in mammalian cells, which likely serves as a signal for the proteasome- mediated DPC processing or other ubiquitin-mediated pathways to facilitate the DPC repair.
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    DNA Replication And Telomere Maintenance Require Pcna-K164 Ubiquitination
    (2020-09) Leung, Wendy
    Genome integrity relies on a robust DNA replication program to ensure faithful duplication of genetic material, free from sequence mutations, deletions or rearrangements. There is an estimated 10 quadrillion (1x1016) cell divisions that occur in the average lifetime of a human being (Weinberg 2014). Thus, cells rely on a global DNA damage response (DDR) network to sense and repair errors that occur during replication to prevent the perpetuation of mutations (Ciccia and Elledge 2010). Although the DDR is highly efficient, some errors may escape repair and interfere with the progression of replication forks. In this scenario, cells utilize DNA damage tolerance (DDT) pathways to bypass errors/lesions encountered during replication and promote replication fork restart (Friedberg 2005, Chang and Cimprich 2009, Ghosal and Chen 2013). A major regulator of DDT pathways is proliferating cell nuclear antigen (PCNA) (Hoege et al. 2002). Ubiquitin modification at the conserved lysine residue 164 (K164) is crucial to DDT pathway choice – mono-ubiquitination activates error-prone translesion synthesis (TLS), while poly-ubiquitination activates error-free template switching (TS) (Shcherbakova and Fijalkowska 2006, Lehmann et al. 2007, Branzei 2011, Sale et al. 2012). However, whether PCNA ubiquitination regulates other genome maintenance mechanisms is unclear. The ends of chromosomes, known as telomeres, are origin-poor and present multiple challenges for the replication machinery including the propensity to form guanine (G)-quadruplexes and RNA-DNA hybrids (Sfeir et al. 2009, Maestroni et al. 2017). Because telomeres are intrinsically “difficult to replicate”, these regions are particularly sensitive to replication stress (Özer and Hickson 2018). In addition to the canonical replication machinery, additional proteins are needed to properly replicate the telomeric duplex. One of these proteins, the TLS polymerase η, functions to alleviate telomeric replication stress (Pope-Varsalona et al. 2014, Garcia-Exposito et al. 2016). The recruitment of TLS polymerases, including Pol η, to DNA lesions occurs through the direct interaction with mono-ubiquitinated PCNA (Bienko et al. 2005). These observations suggest a direct role for PCNA ubiquitination in the replication of telomeres. However, several reports have suggested that TLS can operate in the absence of PCNA ubiquitination (Haracska et al. 2006, Acharya et al. 2007, Parker et al. 2007, Edmunds et al. 2008, Nikolaishvili-Feinberg et al. 2008, Hendel et al. 2011, Krijger et al. 2011), thus it is not clear whether this modification is involved in telomere maintenance. While the role of PCNA-K164 ubiquitination for normal DNA replication and DDT pathway activation has been extensively studied in model systems of yeast, chicken, and mouse, how this modification functions in maintaining human genome stability is still not understood. This thesis addresses several critical functions of K164 ubiquitination in human cells. Studies in PCNAK164R mutants reveal that PCNA ubiquitination is required for gap-filling on the lagging strand behind progressing replication forks (Thakar et al. 2020). Additionally, we provide evidence that K164 ubiquitination functions to resolve late replicating intermediates (LRIs) through mitotic DNA synthesis (MiDAS) and promote efficient origin licensing in the subsequent G1 phase. Finally, we find that post-translational modification of PCNA at K164 regulates telomere maintenance specifically in transformed cells. Together, these studies show that the functions of PCNA-K164 go well beyond progressive DNA synthesis and DDT activation and extend to MiDAS and telomere maintenance.
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    DNA-Protein Cross-links: Formation, Repair, and Inhibition of DNA replication
    (2020-12) Thomforde, Jenna
    DNA-protein crosslinks (DPCs) are ubiquitous DNA lesions that form when cellular proteins become trapped on DNA following exposure to UV light, free radicals, aldehydes, and transition metals. These ultra-bulky lesions are known to disrupt regular DNA cellular machinery, such as replication, transcription, and repair, leading to mutagenesis and carcinogenesis. DPCs can also form endogenously when naturally occurring epigenetic marks (5-formyl cytosine, 5fC) in DNA react with lysine and arginine residues of histones H2A, H3, and H4 to form Schiff base conjugates. However, the understanding of cellular effects on DPCs is not fully understood. The main objective of this thesis was to investigate the effects of DPCs on replication, as well as elucidate mechanisms of repair.In Chapter II, we investigated the local DNA sequence effects on TLS polymerase bypass of 5fC-mediated DNA-peptide crosslinks. Our previous studies revealed that full size DPCs inhibit DNA replication and transcription but can undergo proteolytic cleavage to produce smaller DNA-peptide conjugates. We have shown that when placed in 5'-CXA-3' sequence context (X=5fC-peptide lesion), DNA-peptide crosslinks can be bypassed by human translesion synthesis (TLS) polymerases ƞ and k in an error-prone manner. However, local nucleotide sequence context can have a large effect on replication bypass of bulky lesions by influencing the geometry of the ternary complex between DNA template, polymerase, and the incoming dNTP. In this chapter, model hydrolytically stable DpCs were prepared by oxime ligation between 5fC in DNA and oxy-lysine containing peptides. Primer extension products were analyzed by gel electrophoresis, and steady state kinetics of dAMP incorporation opposite the DpC lesion in different base sequence contexts was investigated. Our results revealed a strong impact of nearest neighbor base identity on polymerase ƞ activity both in the absence and presence of a DpC lesion. Molecular modeling and molecular dynamics simulations of the hPol η ternary complex, containing the DNA template-primer strands with incoming dATP opposite DpC or unmodified C explained structurally how the nature of the 5' and 3' neighbors of this template profoundly impacted its alignment in the C-A mismatch. Our results reveal an important role of base sequence context in promoting TLS related mutational hotspots both in the presence and in the absence of DpC lesions. In Chapter III, we investigated the role of replicative DNA polymerases δ and ε in DpC lesion bypass. TLS polymerase switches are known to be the primary mechanism to bypass bulky DNA lesions such as DNA-peptide crosslinks, however, DpC-containing plasmids were still replicated at relatively high efficiency in TLS-deficient cell lines, leading to the hypothesis that replicative polymerases are also involved in lesion bypass, in a minor role. In Chapter IV, we employed a sensitive nanoLC-ESI+-LC-MS/MS assay to investigate the formation of ROS-induced DPCs between thymidine in DNA and tyrosine in proteins. This methodology was used to analyze the role of metalloprotease Spartan in repair of ROS-induced DPCs in cells and mouse tissues. A 1.5-2 fold increase of thymidine-tyrosine adducts were detected in the brain, heart, livers, and kidneys of Spartan hypomorphic (SPRTNf/-) mice compared to wild type (SPRTN+/+), providing evidence that Spartan plays a direct role in the repair of ROS-induced DPCs.
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    Investigation of the function and control of Dia2, a regulator of genomic stability in budding yeast.
    (2009-11) Kile, Andrew Craig
    Maintenance of genomic integrity can be particularly challenged during DNA replication, which is critical for cellular viability and proliferation. Cancer cells exhibit loss of genomic integrity, thus it is critical to understand the pathways involved in genome maintenance. We have identified the F-box protein Dia2 as a novel and previously unappreciated mediator of genome stability. F-box proteins are substrate specificity subunits of SCF ubiquitin ligases for ubiquitin mediated proteolysis, although most remain uncharacterized in their function or targets. Deletion of the DIA2 gene in Saccharomyces cerevisiae leads to genomic integrity defects, and the Dia2 protein associates with chromatin and origins of replication, indicating it performs a chromatin-associated role in DNA replication. Interestingly, the Yra1 protein was identified to physically interact with Dia2 and promotes Dia2 binding to replication origins yet is not a proteolytic substrate of SCF-Dia2. The Dia2 protein itself is subject to proteolysis, but is stabilized by the activation of the replication checkpoint and this suggests it plays a role during periods of replication stress and DNA damage during S phase. Surprisingly, Dia2 turnover is not controlled by an autocatalytic mechanism involving its F-box domain, but instead relies on a region upstream of its F-box that controls both its stability and nuclear localization. Replication checkpoint activation leads to inhibition of late-firing origins, stabilization of replication forks, as well as stabilization of the Dia2 protein. Our observations indicate that SCF-Dia2 activity performs ubiquitin ligase activity at one or both of these sites that are regulated by the checkpoint. These studies establish a novel link between DNA replication and genomic integrity to the SCF ubiquitin ligase via Dia2.
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    Postreplicative repair is an integral component of lagging strand DNA replication and a suppressor of replication stress
    (2016-04) Becker, Jordan
    Cell division is a basic requirement for the propagation of all organisms. This process begins with a parental cell which divides, leaving two daughter cells. Prior to division, it is necessary for the parental cell to generate a precise duplicate of its genetic material via the process of DNA replication, so that each resulting daughter segregates with a full genetic complement. Errors that occur during this process are thus inherited as mutations by the daughter cells and perpetuated in each subsequent generation along that lineage. Because an estimated 10,000 trillion cell divisions occur in the average lifetime of a human being, it is imperative that this process occurs with a minimum of errors [Quammen 2008]. In the event of difficulty or error, a network of repair and checkpoint pathways has arisen to facilitate the completion of replication with a minimum of inherited mutations [Myung et al. 2001]. The high level of conservation in these replication, repair and checkpoint pathways has allowed us to utilize relatively simple model organisms, such as S. cerevisiae (budding yeast), to better understand how these processes are carried out in more complex metazoan systems. My research has focused on one such group of pathways collectively referred to as postreplicative repair or “PRR” [Chen et al. 2011]. PRR is activated in response to a variety of stressors, which cause difficulty for the replication program and mitigates their impact on genome integrity. The findings included in this dissertation expand our knowledge of stressors, which impact the usage of PRR pathways and moreover describe PRR as an integral component of lagging strand DNA replication.