Browsing by Subject "DNA repair"

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Ancient and Recent Adaptive Evolution of Primate Non-Homologous End Joining Genes
(Public Library of Science, 2010-10-21) Demogines, Ann; East, Alysia M.; Lee, Ji-Hoon; Grossman, Sharon R.; Sabeti, Pardis C.; Paull, Tanya T.; Sawyer, Sara L.
In human cells, DNA double-strand breaks are repaired primarily by the non-homologous end joining (NHEJ) pathway. Given their critical nature, we expected NHEJ proteins to be evolutionarily conserved, with relatively little sequence change over time. Here, we report that while critical domains of these proteins are conserved as expected, the sequence of NHEJ proteins has also been shaped by recurrent positive selection, leading to rapid sequence evolution in other protein domains. In order to characterize the molecular evolution of the human NHEJ pathway, we generated large simian primate sequence datasets for NHEJ genes. Codon-based models of gene evolution yielded statistical support for the recurrent positive selection of five NHEJ genes during primate evolution: XRCC4, NBS1, Artemis, POLλ, and CtIP. Analysis of human polymorphism data using the composite of multiple signals (CMS) test revealed that XRCC4 has also been subjected to positive selection in modern humans. Crystal structures are available for XRCC4, Nbs1, and Polλ; and residues under positive selection fall exclusively on the surfaces of these proteins. Despite the positive selection of such residues, biochemical experiments with variants of one positively selected site in Nbs1 confirm that functions necessary for DNA repair and checkpoint signaling have been conserved. However, many viruses interact with the proteins of the NHEJ pathway as part of their infectious lifecycle. We propose that an ongoing evolutionary arms race between viruses and NHEJ genes may be driving the surprisingly rapid evolution of these critical genes.
Characterization of biological functions of human RNA-binding proteins in Poly(ADP-ribose) polymerase-1-regulated pathways
(2022-07-29) Lin, Wen-Ling (Ph. D. in biochemistry); Liu, Hung-wen, 1952-; Russell, Rick; Lee, Seongmin; Paull, Tanya T.; Fast, Walter L.
Poly(ADP-ribosy)lation (PARylation) can served as a scaffold for noncovalent interactions with various RNA binding proteins including helicases and ribosomal proteins, but many important questions remains regarding the molecular functions and biological roles through the interaction. Based on live-cell imaging assay, I found that DEAD-box helicase 18 (DDX18) and ribosomal protein S19 (RPS19) accumulated to DNA damage sites via PARP-1 activation, indicating their participation in PARP-1-dependent DNA damage repair. My results show that PARP-1 can mediate the association of DDX18 with R-loops thereby modulating R-loop homeostasis and R-loop-dependent DNA damage. DDX18 knockdown renders cells more sensitive to DNA damaging reagents. Furthermore, knockdown of DDX18 reduces R-loop-induced RPA32 and RAD51 foci formation in response to irradiation, and DDX18 depletion also leads to R-loop-induced γH2AX accumulation and genome instability. In addition, DDX18 knockdown abolishes DNA replication due to R-loop accumulation. Taken together, the data uncover new functions of PARP-1 mediated DDX18 in R-loop-mediated events. Here, I also identify RPS19 as a new regulator in DNA double-strand break (DSB) repair. RPS19 was recruited to the DNA damage regions through its putative PAR-binding motif, and the accumulation of RPS19 was PARP-1 dependent. In the absence of RPS19, cells showed reduced RAD51 foci formation, whereas γH2AX and RPA32 foci were increased. Furthermore, the accumulation of RPS19 at DNA damage sites were abolished during the treatment of ATR inhibitors but not ATM inhibitors. Upon X-ray irradiation, RPS19-knockdown cells also decreased phosphorylation of Chk1 at Ser345, which is the downstream of ATR. The results suggest the novel role of RPS19 in ATR pathway during DNA damage repair. In summary, these observations provide a further mechanistic understanding of RNA-binding proteins in PARP-1 mediated DNA damage responses.
Characterization of the mechanisms of ATM activation by the MRN complex and DNA
(2005) Lee, Ji Hoon; Paull, Tanya T.
The complex containing the Mre11, Rad50, and Nbs1 proteins (MRN) is essential for the cellular response to DNA double-strand breaks, integrating DNA repair with the activation of checkpoint signaling through the protein kinase ATM (ataxia Telangiectasia mutated). The ATM kinase signals the presence of DNA double-strand breaks in mammalian cells by phosphorylating proteins which initiate cell cycle arrest or apoptosis. We demonstrate that MRN stimulates the kinase activity of ATM in vitro toward its substrates p53, Chk2, and histone H2AX. We also show that the MRN complex acts as the double-strand break sensor for ATM and recruits ATM to broken DNA molecules. Inactive ATM dimers can be activated in vitro with DNA in the presence of MRN, leading to phosphorylation of downstream targets p53 and Chk2. ATM dimers are dissociated into monomers by MRN in a process that does not require ATM autophosphorylation. Unwinding of DNA ends by MRN is required for ATM stimulation, consistent with the central role of single-stranded DNA as an evolutionary conserved signal for DNA damage.
Consequences, repair, and utilization of an induced double-strand break in the chloroplast DNA of Arabidopsis and tobacco
(2011-05) Kwon, Taegun; Herrin, David L.; Herrin, David L.; Roux, Stanley J.; Jansen, Robert K.; Huq, Enamul; De Lozanne, Arturo; Somanchi, Aravind
In mature chloroplasts, the DNA (cpDNA) is surrounded by a potentially genotoxic environment that would make the mitochondrial DNA milieu look like a “nadree” (picnic). And yet, the slower evolution of cpDNA compared to other cellular genomes suggests that this organelle must have efficient mechanisms for repairing DNA. Unfortunately, those mechanisms have been barely noted, much less studied. This dissertation describes a novel approach that was developed to study how chloroplasts of Arabidopsis repair the most severe form of DNA damage, a double-strand break (described in Chapter 2). The success with this approach also prompted the development of a new method for site-specific modification of tobacco cpDNA that is described in Chapter 3. To study the consequences and repair of a break in the circular plastid genome, we developed an inducible system based on a psbA-intron endonuclease from Chlamydomonas (I-CreII) that specifically cleaves the psbA gene of Arabidopsis. The protein was targeted to the chloroplast using the rbcS1 transit peptide, and activation of the nuclear gene was made dependent on an exogenous inducer (β-estradiol). In Chlamydomonas, I-CreII cleavage at psbA was repaired, in the absence of the intron, by homologous recombination between repeated sequences (20-60 bp) that are abundant in that genome. By comparison, Arabidopsis cpDNA is very repeat-poor. Nonetheless, phenotypically strong and weak transgenic lines were obtained, and shown to correlate with I-CreII expression levels. Southern blot hybridizations indicated a substantial loss of psbA, but not cpDNA as a whole, in the strongly-expressing line. PCR analysis identified deletions nested around the I-CreII cleavage site that were indicative of repair using microhomology (6-12 bp perfect repeats, or 10-16 bp with mismatches) or no homology. The results provide evidence of alternative repair pathways in the Arabidopsis chloroplast that resemble the nuclear microhomology-mediated and nonhomologous end-joining pathways, in terms of the homology requirement. Moreover, when taken together with the results from Chlamydomonas, plus other considerations, the data suggests that an evolutionary relationship may exist between the repeat structure of cpDNA and the organelle’s ability to repair broken chromosomes. Taking advantage of the inducible I-CreII system, I developed a method to delete defined regions of cpDNA in tobacco, which was named DREEM (for direct repeat and endonuclease mediated). Chloroplast transformation was used to introduce an I-CreII cleavage site adjacent to an aadA:gfp marker and flanked by a direct repeat of 84 bp. When chloroplast-targeted I-CreII was induced with β-estradiol during germination, complete loss of the aadA:gfp marker occurred by SSA-type repair involving the 84-bp direct repeat. I obtained additional evidence for DREEM effectiveness by deleting 3.5 kb of native cpDNA that included part of the large ycf1 gene. DREEM can be used for other modifications besides gene deletions, partly because it is seamless and leaves no trace of introduced DNA. Since expression of the endonuclease is controlled by steroid application (and concentration), and the deleted cpDNA is probably destroyed during the SSA process, this inducible gene-ablation technique could enable the study of essential chloroplast genes in vivo.
DNA damage recognition in lesion repair and tolerance
(2016-08-02) Ouzon, Hala; Lee, Seongmin; Fast, Walter L; Hoffman, David W; Kerwin, Sean M; Whitman, Christian P
DNA is constantly attacked by endogenous and exogenous damaging factors to generate a wide variety of DNA lesions that are repaired by damage-specific repair machineries such as base excision DNA repair (BER) and nucleotide excision repair. This work employs kinetic and structural approaches on models of these repair machineries to understand their specificity, explore details of their repair intermediates, and understand how some cells tolerate the purposeful damage of DNA by drugs used in cancer treatment. BER machinery involves DNA glycosylases, AP endonuclease, DNA polymerase and ligase, which identifies, excises, and replaces a lesion. In particular, the cytosine-phosphate-guanine (CpG)-specific DNA glycosylase methyl CpG binding domain 4 (MBD4), initiates the BER pathway via the CpG sites-specific excision of the deamination products of cytosine, 5-methylcytosine, and 5-hydroxy-methylcytosine. The mechanism by which MBD4 recognizes and excises its specific mismatch substrates among the vast excess of correct normal base-pairing is poorly understood. To gain insight into the mechanism, we evaluated the glycosylase activity of MBD4 catalytic domain (MBD4 [superscript cat]) toward various mismatches, conducted MBD4 [superscript cat] mutation studies, and solved four co-crystal structures of MBD4 [superscript cat] in complex with mismatch-containing DNA. Our data suggest that DNA glycosylases can identify these lesions via an intrahelical recognition mechanism. DNA polymerase β (Pol β) fills short nucleotide gaps generated during BER pathway. We describe kinetic and structural studies of a previously unreported intermediate of BER in which Pol β introduces a single nucleotide flap in a sequence-dependent manner. Purposeful DNA damage has been used as an important strategy in fighting cancers, but DNA repair and cellular resistance mechanisms have hindered such efforts. Cisplatin and oxaliplatin are platinum-based chemotherapeutic drugs that do not exhibit cross-resistance. We employ this complementarity to elucidate the cellular resistance mechanism. Our kinetic and structural approaches show that the translesion synthesis DNA polymerase η (Pol η) discriminates between oxaliplatin- and cisplatin-induced DNA damage predominantly at the second base polymerization stage.
Effect of nucleotide binding on Rad50 conformational state, multimeric state, and DNA binding ability
(2009-05) Estrin, Eric; Tanya T. Paull
The ability of an organism to swiftly repair double-stranded breaks (DSBs) in DNA is a crucial process that, if absent, can result in genomic instability. The Mre11/Rad50 protein complex is highly conserved and plays a key role in sensing, processing, and repairing DNA DSBs. Rad50 is known to be necessary for the DNA end-bridging catalyzed by the MRN complex, and association of the broken DNA ends is essential to prevent loss of chromosome fragments in vivo. The Rad50 protein contains a large coiled-coil domain and ATP-binding motifs, with an overall structure similar to the Structural Maintenance of Chromosomes (SMC) family of proteins. Rad50 undergoes ATP-dependent homodimerization, which creates a potential DNA binding cleft. Recently, Rad50 has been shown to have adenylate kinase activity in addition to the previously known ATPase activity. It is still unknown what role ATP-induced dimerization and conformational changes play in pfRad50’s various activities. We hypothesize that dimerization is needed for DNA binding, and that Rad50 requires large conformational changes for proper function in its part in pfMR exonuclease activity. Here, we use site-directed mutagenesis to create Rad50 mutants that have cysteines placed in structurally relevant portions of the protein. With these cysteines, we used disulfide crosslinking, fluorescence, and Fluorescence Resonance Energy Transfer (FRET) to detect if changes in conformation or multimeric state of Rad50 are necessary for adenylate kinase activity, ATPase activity, and DNA binding.
Elucidation of non-B DNA-induced mutagenesis mechanisms : DNA repair proteins are required for the processing of H-DNA and Z-DNA in eukaryotes
(2016-08) McKinney, Jennifer Andrea; Vasquez, Karen M.; DiGiovanni, John; Finch, Rick; Mills, Edward; Kerwin, Sean
The vast majority of all cancers result from some form of genetic instability, thus it is important to study the mechanisms involved. The integrity of DNA can be influenced by secondary structure, and DNA can adopt alternative structures that do not conform to the Watson-Crick B-DNA helix (i.e. non-B DNA). To date, >12 different types of non-B DNA structures have been described including H-DNA and Z-DNA, and these structure-forming sequences are abundant in the human genome, occurring 1/3,000 and 1/50,000 base-pairs for Z-DNA and H-DNA, respectively. Non-B DNA can alter DNA metabolism and contribute to the development of many human diseases. Specific to this project, translocations occurring in the c-MYC and BCL-2 genes, which are shown to contain non-B DNA-forming sequences at translocation breakpoint “hotspots”, are characteristic of certain leukemias and lymphomas. However, the mechanism(s) involved in this process remains undefined. Previously, we found these structures to be mutagenic in bacteria, human cells, and mice, largely by stimulating the formation of DNA double strand breaks (DSBs). We speculated that the helical distortions produced by non-B DNA may be recognized as “damage” by the cell, eliciting an error-prone repair response, resulting in genomic instability. Using genetic-based mutation-reporter assays, we have shown for the first time that these structures are mutagenic in yeast. Furthermore, we have identified DNA repair proteins from both the nucleotide excision repair (NER) and mismatch repair (MMR) pathways to be involved in H-DNA and Z-DNA-induced mutagenesis via distinct mechanisms in both yeast and human cells. We further characterized the functions of these proteins using biochemical and molecular biology assays and found that they are enriched at sites of H-DNA and/or Z-DNA, and have cleavage activity at or near the structure. Taken together, these results suggest that non-B structures are processed in an error-prone fashion via various novel structure-specific repair pathways in which repair proteins from multiple pathways cooperate. The results obtained have enhanced our knowledge of DNA structure-induced genetic instability in disease etiology, and will guide future studies in the development of novel strategies to treat and/or prevent genetic diseases.
Eukaryotic transcriptional regulation : from data mining to transcriptional profiling
(2008-12) Morgan, Xochitl Chamorro; Iyer, Vishwanath R.
Survival of cells and organisms requires that each of thousands of genes is expressed at the correct time in development, in the correct tissue, and under the correct conditions. Transcription is the primary point of gene regulation. Genes are activated and repressed by transcription factors, which are proteins that become active through signaling, bind, sometimes cooperatively, to regulatory regions of DNA, and interact with other proteins such as chromatin remodelers. Yeast has nearly six thousand genes, several hundred of which are transcription factors; transcription factors comprise around 2000 of the 22,000 genes in the human genome. When and how these transcription factors are activated, as well as which subsets of genes they regulate, is a current, active area of research essential to understanding the transcriptional regulatory programs of organisms. We approached this problem in two divergent ways: first, an in silico study of human transcription factor combinations, and second, an experimental study of the transcriptional response of yeast mutants deficient in DNA repair. First, in order to better understand the combinatorial nature of transcription factor binding, we developed a data mining approach to assess whether transcription factors whose binding motifs were frequently proximal in the human genome were more likely to interact. We found many instances in the literature in which over-represented transcription factor pairs co-regulated the same gene, so we used co-citation to assess the utility of this method on a larger scale. We determined that over-represented pairs were more likely to be co-cited than would be expected by chance. Because proper repair of DNA is an essential and highly-conserved process in all eukaryotes, we next used cDNA microarrays to measure differentially expressed genes in eighteen yeast deletion strains with sensitivity to the DNA cross-linking agent methyl methane sulfonate (MMS); many of these mutants were transcription factors or DNA-binding proteins. Combining this data with tools such as chromatin immunoprecipitation, gene ontology analysis, expression profile similarity, and motif analysis allowed us to propose a model for the roles of Iki3 and of YML081W, a poorly-characterized gene, in DNA repair.
Homeodomain proteins directly regulate ATM kinase activity
(2018-02-07) Johnson, Tanya Ellen; Paull, Tanya T.; Huibregtse, Jon; Lambowitz, Alan; Russell, Rick; Iyer, Vishy
The ataxia-telangiectasia mutated (ATM) kinase is a master regulator involved in the detection and repair of DNA double-strand breaks (DSBs). The Mre11/Rad50/Nbs1 (MRN) complex recruits and activates ATM at DSBs, causing a signaling cascade to initiate cell-cycle checkpoint arrest, DNA repair, and apoptosis. Alternatively, ATM can be activated by direct oxidation and may act as a redox sensor in the cellular response to oxidative stress. Loss of functional ATM results in ataxia-telangiectasia, a genomic instability disorder characterized by neurodegeneration, DNA repair defects, and predisposition to cancer. Understanding how ATM kinase activity is regulated is critical to understanding its function in the DNA damage and oxidative stress responses. Recently, ATM kinase activity was shown to be stimulated directly by a homeodomain protein, NKX3.1, in prostate cells. NKX3.1 is thought to be the gatekeeper to prostate tumor suppression and, although normally expressed solely in prostate cells, as many as 80% of prostate tumors exhibit loss of NKX3.1 protein. One mechanism of tumor suppression by NKX3.1 may be modulating the DNA damage response via ATM. In in vitro kinase assays, NKX3.1 protein was sufficient to stimulate ATM both in the MRN-mediated and oxidative pathways. Thus, NKX3.1 acts as a tissue-specific modulator of ATM function. Since the conserved homeodomain was critical for the stimulation of ATM by NKX3.1, it poses the question whether this is a general mechanism of ATM regulation by all homeodomain proteins. Here, I address this question by identifying other tissue-specific regulators of ATM activity within the homeodomain family of proteins. Five homeodomain proteins (TTF1, HOXB7, NKX2.5, NKX2.2, and CDX2) were tested for the ability to regulate ATM activity. In in vitro kinase assays, all of the homeodomain proteins tested stimulated ATM kinase activity to varying degrees, suggesting that the homeodomain itself may act as a conserved regulator of ATM function. I show that CDX2 modulates ATM function in mammalian cell lines with global effects on the DNA damage response. Together, these data support the hypothesis that homeodomain proteins regulate ATM function in a tissue-specific manner.
Identification and characterization of a positive regulatory region for activation induced cytidine deaminase mediated gene conversion in chicken B cells
(2009-12) Kim, Yonghwan, 1975-; Tian, Ming, Ph. D.; Tucker, Philip W.; Paull, Tanya T.; Iyer, Vishwanath; Yin, Whitney
B cells have unique machinery to make up a large pool of antibody repertoire. After V(D)J recombination in early B cell development, the rearranged immunoglobulin genes are further diversified by somatic hypermutation (SHM), gene conversion (GC) and class switch recombination (CSR). Acitvation induced cytidine deaminase (AID) is a key initiating factor for SHM, GC and CSR. A majority of research data supports the model that AID modifies Ig genes at the DNA level by deaminating cytosines to uracils. The mutagenic activity of AID is largely restricted to Ig genes to avoid genomic instability in general. The specificity cannot be attributed to the primary sequence of the Ig genes since unrelated DNA is mutated by AID in the context of Ig genes. A clue to this problem is that AID function is dependent on transcription. Since not all transcribed genes are mutated by AID, there must be something special about the transcription of Ig genes, and the reasoning has prompted extensive analysis of Ig promoters and enhancers. We addressed this question in chicken B cell line DT40. We identified a 2.4-kilobase regulatory region which is important for AID function both within and outside of Ig locus. This regulatory region contains binding sites for multiple transcription factors. Mutation of these binding sites impairs AID mediated gene conversion. In addition, ablation of NF-κB family member, c-Rel and p50, reduces the AID targeting function of this regulatory region. Since the implicated transcription factors have been reported to associate with histone acetylases, the regulatory region may function by facilitating the access of AID to target DNA. To test this hypothesis, we used the I-SceI endonuclease and dam methylase as probes for chromatin structure. We found that the regulatory region does not increase chromatin accessibility to these probes. In fact, the regulatory region appears to interfere with the cleavage of target DNA by I-SceI. Another possible role of the regulatory region could be direct recruitment of AID to Ig genes. To test this hypothesis, we utilized Dam identification method. Surprisingly, we found that the regulatory region facilitates AID targeting to the Igλ locus.
Investigation of the mechanistic basis for the role of Rad50 in double-strand break repair
(2006) Bhaskara, Venugopal; Paull, Tanya T.
Members of the Rad52 epistasis group, which includes a heterotrimeric complex, formed by Mre11, Rad50 and Nbs1 (Xrs2 in yeast) helps in the protection of cell’s genetic content from DNA doublestrand breaks. The Rad50 component of the human Mre11/Rad50/Nbs1 (Xrs2 in yeast) complex (MRN(X)) belongs to the ABC superfamily of ATPases and is conserved among all organisms and contains Walker A (N-Terminus) and Walker B (C-Terminus) ATPase domains connected by a long coiled-coil region. We show for the first time that Rad50 shows adenylate kinase activity (ATP + AMP ↔ 2ADP) and that this activity is important for tethering of DNA ends. We further show that Rad50 can catalyze “reverse” adenylate kinase activity (2ADP → ATP + AMP) and this activity is stimulated in the presence of linear DNA ends. We also show that the signature motif of Rad50 is essential for all ATP-dependent activities in vivo and in vitro.
Mechanisms of target search by DNA-binding proteins
(2018-02-28) Brown, Maxwell William; Finkelstein, Ilya J.; Paull, Tanya; Russell, Rick; Dalby, Kevin; Taylor, David
All genetic information is preserved within the DNA duplex. For successful propagation of this genetic code, DNA must be accurately replicated, repaired, and transcribed. These fundamental processes require DNA-binding proteins that recognize specific DNA sequences or structures. These proteins must quickly find a specific target amidst a vast excess of non-specific, yet structurally similar, DNA. Moreover, target search must proceed in the context of nucleosomes, transcription factors, and other protein roadblocks. Uncovering how proteins locate their targets is important for understanding key regulatory mechanisms involved in nearly all genomic transactions. My research aims to understand the mechanisms and structural motifs that regulate target search. Our approach uses single-molecule microscopy to visualize this multi-step and dynamic process in real-time. My first project studied the heterodimeric DNA MisMatch Repair (MMR) complex Msh2-Msh3. Msh2-Msh3 recognizes DNA lesions implicated in MMR, Single-Strand DNA Anealing (SSA), and TriNucleotide Repeat (TNR) expansion disorders. My work provided the first comprehensive characterization of how Msh2-Msh3 can ‘hop’ over nucleosomes via the Msh3 Mispair Binding Domain (MBD) as it searches for DNA lesions (Chapter 2). I then focused on the type I CRISPR-Cas system, which confers bacteria and archaea with immunity against foreign DNA. This process is initiated by the sequence-specific targeting of foreign DNA by Cascade; a multi-subunit, RNA-guided, target recognition complex. I discovered that sequence and structural conservation of basic residues in Cascade regulates the diffusion of the complex on non-specific DNA. I then observed the Cascade mediated recruitment of downstream proteins Cas3 and Cas1-Cas2, culminating in the formation of a Primed Acquisition Complex (PAC) which I conjecture is required for cells to respond to mutated DNA targets (Chapter 4). Furthermore, I developed new methods relating to single-molecule microscopy, including a high-throughput method to make DNA curtains, a fluorescent labeling strategy for conjugating dyes to proteins, and a high-throughput force-based assay (Chapter 3). In addition to providing protocols and tools that will be useful for future single-molecule studies, my research has made key contributions to the MMR and CRISPR fields by shedding light on mechanisms that contribute to target search and roadblock removal/avoidance on DNA.
Mu Insertions Are Repaired by the Double-Strand Break Repair Pathway of Escherichia coli
(Public Library of Science, 2012-04-12) Jang, Sooin; Sandler, Steven J.; Harshey, Rasika M.
Mu is both a transposable element and a temperate bacteriophage. During lytic growth, it amplifies its genome by replicative transposition. During infection, it integrates into the Escherichia coli chromosome through a mechanism not requiring extensive DNA replication. In the latter pathway, the transposition intermediate is repaired by transposase-mediated resecting of the 5′ flaps attached to the ends of the incoming Mu genome, followed by filling the remaining 5 bp gaps at each end of the Mu insertion. It is widely assumed that the gaps are repaired by a gap-filling host polymerase. Using the E. coli Keio Collection to screen for mutants defective in recovery of stable Mu insertions, we show in this study that the gaps are repaired by the machinery responsible for the repair of double-strand breaks in E. coli—the replication restart proteins PriA-DnaT and homologous recombination proteins RecABC. We discuss alternate models for recombinational repair of the Mu gaps.
Mutagenicity and repair of small DNA lesions
(2023-08-07) Schmaltz, Lillian Fei Gang; Lee, Seongmin; Fast, Walter; Vasquez, Karen; Yang, Kun
DNA is continuously attacked by both endogenous and exogenous damaging agents. The resulting DNA damage can play a significant role in mutagenesis and carcinogenesis. Though cells can experience up to 10⁵ DNA damaging events per day, they employ mechanisms including, base excision repair (BER) and translesion synthesis (TLS) to repair and bypass the DNA lesions, respectively. Evaluating the mechanisms by which cells respond to DNA damage provides insights to whether these lesions, though small and non-distorting, may be pro-mutagenic and lead to disease. Oxidative stress is ubiquitous and results in two major products, 8oxoA and 8oxoG. Using a supF-based mutation assay, we observed that the DNA lesion, 8oxoA, at frequencies as high as 19% in bacterial cells. Additionally, the mutational spectrum revealed the majority of mutations induced were A to C transversions supporting our previous data that dGTP will preferentially be inserted opposite oxoA. To investigate the repair of such oxidative damage, we solved the structure of 2’F-8oxoG in complex with a human oxoG DNA glycosylase (hOGG1). Use of the 2’F inhibited cleavage of the glycosidic bond and allowed us to visualize the DNA lesion in the active site of a catalytically competent glycosylase. Using this same supF assay, we evaluated the mutational frequencies 2’F-N7meG and its ring-opened product, 2’F-N7meFapyG. While replication across N7meFapyG was mutagenic, N7meG was not and our structure of N7meG with TLS polymerase, Dpo4, provided insights to how cells can accurately replicate across this DNA lesion. Lastly, we investigated the repair of DNA mismatches by Methyl-CpG-Binding Domain 4 (MBD4). Our structural and kinetic analysis highlights the significance of the elusive arginine finger in the recognition of a T:G mismatch.
The P. furiosus Mre11/Rad50 complex facilitates 5’ strand resection by the HerA helicase and NurA nuclease at a DNA double-strand break
(2010-05) Hopkins, Ben Barrett; Paull, Tanya T.; Dudley, Jaquelin P.; Graham, David E.; Jayaram, Makkuni; Yin, Whitney Y.
The Mre11/Rad50 complex has been implicated in the early steps of DNA double-strand break (DSB) repair through homologous recombination in several organisms. However, the enzymatic properties of this complex are incompatible with the generation of 3’ single-stranded DNA for recombinase loading and strand exchange. In thermophilic Archaea, the mre11 and rad50 genes cluster in an operon with genes encoding a bidirectional DNA helicase, HerA, and a 5’ to 3’ exonuclease, NurA, suggesting these four enzymes function in a common pathway. I show that purified Mre11 and Rad50 from Pyrococcus furiosus act cooperatively with HerA and NurA to resect the 5’ strand at a DNA end under physiological conditions in vitro where HerA and NurA alone do not show detectable activity. Furthermore, I demonstrate that HerA and NurA physically interact, and this interaction stimulates both helicase and nuclease activities. The products of HerA/NurA long-range resection are oligonucleotide products and HerA/NurA activity demonstrates both sequence specificity and a preference to cut at a specific distance from the DNA end. I demonstrate a novel activity of Mre11/Rad50 to make an endonucleolytic cut on the 5’ strand, which is consistent with a role for the Mre11 nuclease in the removal of 5’ protein conjugates. I also show that Mre11/Rad50 stimulates HerA/NurA-mediated resection through two different mechanisms. The first involves an initial Mre11 nucleolytic processing event of the DNA to generate a 3’ ssDNA overhang, which is then resected by HerA/NurA in the absence of Mre11/Rad50. The second mechanism likely involves local unwinding of the DNA end in a process dependent on Rad50 ATPase activity. I propose that this unwinding step facilitates binding of HerA/NurA to the DNA end and efficient resection of the break. Furthermore, the binding affinity of NurA for 3’ overhang and unwound DNA end substrates partially explains the efficiency of the two resection mechanisms. Lastly, 3’ single-stranded DNA generated by these enzymes can be used by the Archaeal RecA homolog RadA to catalyze strand exchange. This work elucidates how the conserved Mre11/Rad50 complex promotes DNA end resection in Archaea, and may serve as a model for DSB processing in eukaryotes.
Regulation of DNA damage response by ATM and DNA-PKcs
(2015-12-09) Zhou, Yi, Ph. D.; Paull, Tanya T.; Finkelstein, Ilya J; Iyer, Vishwanath R; Miller, Kyle M; Lee, Seongmin
The 5’ strand resection of DNA double strand breaks (DSBs) initiates homologous recombination (HR) and is critical for genomic stability. To date there is no quantitative method to measure single-stranded DNA (ssDNA) intermediates of resection in mammalian cells. In this study I develop a quantitative PCR (qPCR)-based assay to quantitate ssDNA intermediates, specifically, the 3’ ssDNA product of resection at specific DSBs induced by AsiSI restriction enzyme in human cells. I protect the large mammalian genome from shearing by embedding the cells in low-gelling-point agar during genomic DNA extraction, and measure the levels of ssDNA intermediates by qPCR following restriction enzyme digestion. This assay is more quantitative and precise compared with existing protein foci-based methods. Using this assay I quantitatively measure ssDNA intermediates of resection in human cells and find that the 5' strand at endonuclease-generated break sites is resected up to 3.5 kb in a cell cycle dependent manner. Depletion of CtIP, Mre11, Exo1, or SOSS1 blocks resection, while depletion of 53BP1, Ku or DNA-dependent protein kinase catalytic subunit (DNA-PKcs) leads to increased resection as measured by this method. While 53BP1 negatively regulates DNA end processing, depletion of BRCA1 does not, suggesting that the role of BRCA1 in HR is primarily to promote RAD51 filament formation, not to regulate end resection. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, I also show that DNA-PKcs, a classic non-homologous end joining (NHEJ) factor, antagonizes DSB resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. ATM kinase activity can compensate for DNA-PKcs autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11/Rad50/Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs autophosphorylation. This work suggests that, in addition to its key role in NHEJ, DNA-PKcs also acts in concert with MRN and ATM to regulate resection and thus DNA repair pathway choice. In addition, I find that MRN strongly suppresses DNA Ligase IV/XRCC4-mediated end rejoining, whereas it dramatically promotes DNA end ligation by the DNA Ligase III/XRCC1 complex. The Ataxia-Telangiectasia mutated (ATM) protein is a key regulator of checkpoint activation and HR in response to DSBs. The MRN complex acts as a DSB sensor and is essential for ATM recruitment to broken DNA ends and ATM activation. However, the precise mechanism for ATM activation upon DNA damage and ATM inactivation after DNA repair has remained poorly understood. Phosphorylation of ATM has been suggested to play important roles in this process. Autophosphorylation of ATM at four sites (S1981, S367, S1893, and S2996) has been shown to be essential for ATM activation and function in response to DNA damage in human cells. However, mutations at these four sites do not affect ATM kinase activity in vitro or in mouse models, suggesting that there are other mechanisms for regulation of ATM activity. Previous studies show that CDK5-mediated phosphorylation of ATM at Ser794 and EGFR-mediated phosphorylation of ATM at Tyr370 both positively regulate ATM activation upon DNA damage. In this study, I further propose that DNA-dependent protein kinase (DNA-PK) negatively regulates ATM activity through phosphorylation of ATM at multiple sites. ATM is hyperactive when DNA-PK activity is blocked by DNA-PK specific inhibitor or when the DNA-PKcs gene is deleted in cells. Pre-incubation of ATM protein with DNA-PK significantly inhibits ATM kinase activity in vitro. Using mass spectrometry analysis and site-directed mutagenesis, I characterized three clusters of sites: S85/T86, T372/T373 and T1985/S1987/S1988. The phospho-mimetic mutations at these residues repress ATM activation both in vitro and in cells. Overexpression of phospho-mimetic ATM mutants in ATM-deficient cells fails to restore cell survival, DSB end resection and G2/M checkpoint activation in response to DNA damage. In addition, I have observed that the phospho-blocking ATM mutants, T1985A/S1987A/S1988A and T86A/T373A, are resistant to DNA-PK pre-incubation in vitro and that overexpression of ATM T1985A/S1987A/S1988A mutant fails to respond to DNA-PK inhibition in comparison to wild-type ATM in cells. Taken together, my data suggests that the NHEJ factor DNA-PK suppresses the catalytic activity of ATM through phosphorylation and that the phosphorylation of a group of Ser/Thr residues on ATM negatively regulates ATM signaling upon DNA damage. Since ATM is known to promote the HR pathway, this may provide a novel mechanism for DNA repair pathway choice upon DNA damage as well as ATM inactivation after DNA repair.
Regulation of the activity of a budding yeast DNA damage repair enzyme Sae2
(2014-12) Fu, Qiong, Ph. D.; Paull, Tanya T.; Iyer, Vishwanath R; Jayaram, Makkuni; Johnson, Arlen W; Zhang, Yan
In response to DNA damage, many repair and signaling molecules mobilize rapidly to the sites of DNA double-strand breaks (DSBs). This network of immediate responses is regulated at the level of post-translational modifications to coordinate DNA repair and checkpoint signaling. Here we investigate the DNA damage-induced oligomeric transitions of the Sae2 protein, an important enzyme in the initiation of DSB repair. Sae2 is a target of multiple phosphorylation events, which we identify and characterize in vivo in budding yeast. Both cell cycle-dependent and DNA damage-induced phosphorylation of Sae2 are important for the cell survival after DNA damage, and the cell cycle-regulated modifications are required to prime the damage-dependent events. We find that Sae2 exists in the form of inactive oligomers that are transiently released into smaller active units by these series of phosphorylation events. DNA damage also triggers removal of Sae2 through autophagy and proteasomal degradation, ensuring that active Sae2 is present only transiently in cells. This analysis provides evidence for a novel type of protein regulation where the activity of an enzyme is controlled dynamically by post-translational modifications that regulate its solubility and oligomeric state. Budding yeast Ess1 is a phosphorylation-specific prolyl isomerase. Its human homolog Pin1 is found to isomerize CtIP, the human functional ortholog of Sae2, and promote the proteasomal degradation of CtIP. However, I could neither detect any interaction between Ess1 and Sae2, nor observe any change in Sae2 protein level while overexpressing wild-type or mutant Ess1, suggesting Ess1 does not act on Sae2, like Pin1 does on CtIP. The increased DNA damage sensitivity of Ess1 mutants indicates that Ess1 is involved in DNA repair, but not related to Sae2. Since Ess1 plays an important role in transcription termination together with a RNA 3’ end processing factor Pcf11, I overexpressed wild-type Pcf11 and found it significantly increased the DNA damage resistance of either wild-type or H164R mutant Ess1 cells, and also the sae2Δ cells. These results imply that Ess1, Pcf11 and Sae2 might contribute to DNA damage repair through transcription termination, which links transcription termination and DNA damage repair together.
Role of bromodomain containing proteins in the DNA damage response
(2016-09-09) Gong, Fade; Miller, Kyle M.; Iyer, Vishwanath R; Paull, Tanya T; Vasquez, Karen M; Xhemalce, Blerta
Chromatin-based DNA damage response (DDR) mechanisms are fundamental for preventing genome and epigenome instability, which are hallmarks of cancer. How chromatin promotes genome-epigenome integrity in response to DNA damage is a critical question. Chromatin acetylation is a key signaling event involved in detecting, signaling and repairing DNA damage. The bromodomain (BRD) containing protein is the primary reader of acetylation. Thus, BRD proteins represent attractive candidates for reading damaged chromatin to mediate genome-epigenome integrity. In the first part of this project, I performed a screen to analyze the dynamics of BRD protein at DNA damage sites. I identified one-third of BRD proteins relocalized upon DNA damage, a phenomenon common to DNA damage factors. In the second part of my thesis work, I functionally studied the BRD protein ZMYND8 in a novel transcription-dependent DNA damage recognition pathway. Upon DNA damage specifically within actively transcribing chromatin, ZMYND8 is recruited through its BRD to TIP60 mediated H4 acetylations. ZMYND8 associates with the NuRD complex and promotes its accumulation at damage sites to facilitate transcriptional repression and promote repair by homologous recombination (HR). To investigate mechanisms regulating this novel ZMYND8-NuRD pathway, I performed another screen to check the recruitment of ZMYND8 interacting factors as well as their effects on ZMYND8 recruitment. I identified the H3K4me3 specific histone demethylase KDM5A is a key upstream regulator of this transcription-dependent DNA damage recognition pathway. Upon DNA damage, KDM5A mediates the removal of H3K4me3 around active chromatin near damage sites, which is an essential step to facilitate recruitment of ZMYND8 and NuRD complex to DNA damage. Similar to ZMYND8 and NuRD, depletion of KDM5A also impairs damage induced transcriptional silencing and DNA double-strand break (DSB) repair by homologous recombination (HR). The DDR is not only a dynamic process focusing that regulates protein factor interaction at DNA damage sites, but also promotes transcriptional changes of some genes upon DNA damage. In another part of this project, I screened the functional role of BRD proteins in regulating transcription in response to different types of damage. I identified two novel p53 target genes SP110 and SP140. In response to treatment with the DNA damaging chemotherapeutic agent, Doxorubicin, in U2OS cells, SP110 and SP140 are upregulated in a p53 dependent manner. In summary, this study provides a comprehensive view for BRD reader proteins in promoting the DDR within acetylated chromatin to preserve genome-epigenome stability.
Single-molecule studies reveal the dynamics of DNA repair and transcription-associated proteins
(2019-01-25) Kim, Yoori; Finkelstein, Ilya J.; Russell, Rick; Vasquez, Karen M; Dalby, Kevin N; Lee, Seongmin
Intrinsically disordered regions (IDR) are protein segments that lack a defined tertiary structure. IDRs are enriched in eukaryotic chromatin-binding proteins, where they modulate protein-DNA and protein-protein interactions. In this thesis, I probe the function(s) of IDRs via two case studies: the yeast mismatch repair (MMR) protein Mlh1- Pms1 and transcription factors (TFs) derived from C. albicans, a pathogenic yeast. Using single-molecule DNA curtain assays, I demonstrate novel roles for IDRs in promoting facilitated diffusion of Mlh1-Pms1 on DNA. IDRs improve Mlh1-Pms1’s ability to bypass a single nucleosome and to navigate dense nucleosome arrays that resemble chromatin. Moreover, these IDRs are critical for the Mlh1-Pms1 ATPase activity and also for nicking of the DNA substrate. I propose that conformational changes in the Mlh1-Pms1 IDRs alter DNA interactions and the nucleolytic activity of neighboring structured domains. I also examine the dynamics of PCNA, another essential MMR factor, in the context of trinucleotide repeat (TNR) instability. I show that Replication Factor C preferentially loads PCNA onto (CAG)₁₃ structures. The (CAG)₁₃ repeat captures the loaded PCNA and prevents PCNA from diffusing. Lastly, I reveal a novel role for IDRs in DNA condensation by studying Efg1, a TF that regulates a cell-type switching network in C. albicans. Efg1 encodes a specific IDR of low complexity, referred to as the prion-like domain (PrLD). I show that the PrLD is critical for the DNA condensation and recruiting other PrLD-containing TFs, wherein nucleosomes regulate the TF-DNA dynamics. I propose a model where transcription factors become concentrated via phase separation and bring gene regulatory elements together to promote gene activation. Overall, this study provides mechanistic insights into the functions of IDRs in the dynamic behavior of DNA-binding proteins
Strategies for deciphering the genome
(2014-12) Lou, Dianne In-Hye; Sawyer, Sara L.; Press, William H; Paull, Tanya T; Sullivan, Christopher S; Ehrlich, Lauren IR; Miller, Kyle M
The development of highly sophisticated technologies has ushered in the era of the genome. Most importantly, high-throughput sequencing technologies has vastly expanded the number of available genome projects of many different organisms. One of challenges that we now face is in understanding the information encoded within these genomes. Within each chapter of this dissertation, information from existing genome projects are used to answer fundamental biological questions related to human disease and an attempt to further advance new technologies is made. In chapter 2, I describe a novel method that decreases the error rates associated with next-generation sequencing technologies, allowing for the investigation of more complex and heterogenous samples relevant to many biological systems. In chapter 3, I use available primate genome projects to understand the evolutionary trajectory of two DNA repair genes, whose defect increases the development of breast and ovarian cancers. Finally, in chapter 4, wild-type primate alleles are used as tools to uncover novel mechanisms in the lifecycle of viruses. Although seemingly non-overlapping, each of these studies is centered around using the sea of information that is now readily available in order to decipher the many secrets encoded by genome.