Biochemical Characterization of DNA Glycosylases from Mycobacterium Tuberculosis

The DNA glycosylases function in the first step of the base excision repair (BER) process, that is responsible for removing base lesions resulting from oxidation, alkylation or deamination. The DNA glycosylases that recognize oxidative base damage fall into two general families: the Fpg/Nei family and the Nth superfamily. Based on protein sequence alignments, we identified four putative Fpg/Nei family members as well as a putative Nth protein in Mycobacterium tuberculosis H37Rv, the causative agent of tuberculosis. While Fpg proteins are widely distributed among the bacteria and plants, Nei homologs are sparsely distributed across phyla, and are only found in γ-proteobacteria, actinobacteria and metazoans. Interestingly, M. tuberculosis H37Rv harbors two proteins (Rv2464c and Rv3297) from the Nei clade and two (Rv2924c and Rv0944) from the Fpg clade. All four Fpg/Nei proteins were successfully overexpressed by using a novel bicistronic vector, which theoretically prevented stable mRNA secondary structure(s) surrounding the translation initiation region (TIR) thereby improving translation efficiency. Additionally, MtuNth (Rv3674c) was also overexpressed in soluble form. The substrate specificities of the purified enzymes were characterized in vitro with oligonucleotide substrates containing single lesions. Some were further characterized by gas chromatography/mass spectrometry (GC/MS) analysis of products released from γ-irradiated DNA. MtuFpg1 (Rv2924c) has a substrate specificity similar to that of EcoFpg and recognizes oxidized purines. Both EcoFpg and MtuFpg1 are more efficient at removing spiroiminodihydantoin (Sp) than 7,8-dihydro-8-oxoguanine (8-oxoG); however, MtuFpg1 has a substantially increased opposite base discrimination compared to EcoFpg. The Rv0944 gene encodes MtuFpg2, which contains only the C-terminal domain of an Fpg protein and has no detectable DNA binding activity or DNA glycosylase/lyase activity and thus appears to be a pseudogene. MtuNei1 (Rv2464c) recognizes oxidized pyrimidines not only on doublestranded DNA but also on single-stranded DNA. It also exhibits uracil DNA glycosylase activity as well as weak activity on FapyA and FapyG. MtuNth recognizes a variety of oxidized bases, such as urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5- OHU), 5-hydroxycytosine (5-OHC) and methylhydantoin (MeHyd) as well as FapyA, FapyG and 8-oxoadenine (8-oxoA). Both MtuNei1 and MtuNth excise thymine glycol (Tg); however, MtuNei1 strongly prefers the (5R) isomers of Tg, whereas MtuNth recognizes only the (5S) isomers. The other Nei paralog, MtuNei2 (Rv3297), did not demonstrate activity in vitro as a recombinant protein, but when expressed in Escherichia coli, the protein decreased the spontaneous mutation frequency of both the fpg mutY nei triple and nei nth double mutants, suggesting that MtuNei2 is functionally active in vivo recognizing both guanine and cytosine oxidation products. The kinetic parameters of the MtuFpg1, MtuNei1 and MtuNth proteins on selected substrates were also determined and compared to those of their E. coli homologs. Since pathogenic bacteria are often exposed to an oxidative environment, such as in macrophages, our data, together with previous observations, support the idea that the BER pathway is of importance in protecting M. tuberculosis against oxidative stress, as has been observed with other pathogens

Mechanisms of Genome Maintenance in Plants: Playing It Safe With Breaks and Bumps

Maintenance of genomic integrity is critical for the perpetuation of all forms of life including humans. Living organisms are constantly exposed to stress from internal metabolic processes and external environmental sources causing damage to the DNA, thereby promoting genomic instability. To counter the deleterious effects of genomic instability, organisms have evolved general and specific DNA damage repair (DDR) pathways that act either independently or mutually to repair the DNA damage. The mechanisms by which various DNA repair pathways are activated have been fairly investigated in model organisms including bacteria, fungi, and mammals; however, very little is known regarding how plants sense and repair DNA damage. Plants being sessile are innately exposed to a wide range of DNA-damaging agents both from biotic and abiotic sources such as ultraviolet rays or metabolic by-products. To escape their harmful effects, plants also harbor highly conserved DDR pathways that share several components with the DDR machinery of other organisms. Maintenance of genomic integrity is key for plant survival due to lack of reserve germline as the derivation of the new plant occurs from the meristem. Untowardly, the accumulation of mutations in the meristem will result in a wide range of genetic abnormalities in new plants affecting plant growth development and crop yield. In this review, we will discuss various DNA repair pathways in plants and describe how the deficiency of each repair pathway affects plant growth and development

Localization of a Microsporidia ADAM (A Disintegrin and Metalloprotease Domain) Protein and Identification of Potential Binding Partners.

Microsporidia are spore-forming, obligate intracellular pathogens typically associated with opportunistic infections in immunocompromised individuals. Treatment options for microsporidia infections in humans are limited and additional research is necessary to create better therapeutic agents. For many pathogenic organisms, adhesion to the host cell surface is a prerequisite for tissue colonization and invasion. Our previous research has demonstrated a direct relationship between adherence of microsporidia spores to the surface of host cells and infectivity in vitro. In an effort to better understand adherence, we have turned our attention to determining what proteins may be involved in this process. Examination of the Encephalitozoon cuniculi genome database revealed a gene encoding a protein with sequence homology to members of the ADAM (a disintegrin and metalloprotease) family of type I transmembrane glycoproteins. The microsporidia ADAM (MADAM) protein is of interest because ADAMs are known to be involved in a variety of biological processes including cell adhesion, proteolysis, cell fusion, and signaling. The objectives for this study were to examine the localization of MADAM, analyze its potential involvement during adherence and/or host cell infection, and to identify potential binding partners or substrates. Through the use of immunoelectron transmission microscopy, we demonstrated that MADAM is localized to the surface exposed exospore, plasma membrane, and the polar sac-anchoring disk complex (a bell-shaped structure at the spore apex involved in the infection process). Location of MADAM within the exospore and polar sac-anchoring disk suggests that MADAM is in a position to facilitate spore adherence or host cell infection. Thus far, we have been unable to conclusively demonstrate that MADAM is involved in either event. Through the use of a yeast two-hybrid system, we were able to identify polar tube protein 3 (PTP3) as a potential binding partner or substrate for the MADAM protein. The interaction between MADAM and PTP3 was confirmed by in vitro co-immunoprecipitation. PTP3 is hypothesized to be involved in the process of polar tube extrusion by stabilizing the interaction between PTP1-PTP2 polymers. Further analysis of the interaction between MADAM and PTP3 may lead to a better understanding of the events that occur during polar tube extrusion

Investigating the Structural Basis for Human Disease: APOBEC3A and Profilin

Analyzing protein tertiary structure is an effective method to understanding protein function. In my thesis study, I aimed to understand how surface features of protein can affect the stability and specificity of enzymes. I focus on 2 proteins that are involved in human disease, Profilin (PFN1) and APOBEC3A (A3A). When these proteins are functioning correctly, PFN1 modulates actin dynamics and A3A inhibits retroviral replication. However, mutations in PFN1 are associated with amyotrophic lateral sclerosis (ALS) while the over expression of A3A are associated with the development of cancer. Currently, the pathological mechanism of PFN1 in this fatal disease is unknown and although it is known that the sequence context for mutating DNA vary among A3s, the mechanism for substrate sequence specificity is not well understood. To understand how the mutations in Profilin could lead to ALS, I solved the structure of WT and 2 ALS-related mutants of PFN1. Our collaborators demonstrated that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization was illuminated by my X-ray crystal structures of several PFN1 proteins. I found an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis. To characterize A3A’s substrate specificity, we solved the structure of apo and bound A3A. I then used a systematic approach to quantify affinity for substrate as a function of sequence context, pH and substrate secondary structure. I found that A3A preferred ssDNA binding motif is T/CTCA/G, and that A3A can bind RNA in a sequence specific manner. The affinity for substrate increased with a decrease in pH. Furthermore, A3A binds tighter to its substrate binding motif when in the loop region of folded nucleic acid compared to a linear sequence. This result suggests that the structure of DNA, and not just its chemical identity, modulates A3 affinity and specificity for substrate

Investigating enzyme communication during base excision repair in Escherichia coli

Mismatch uracil DNA Glycosylase (MUG) from Escherichia coli is an initiating enzyme in the base excision repair (BER) pathway and is responsible for the removal of 3,N4-ethenocytosine and uracil from DNA during the stationary phase of E.coli cell growth. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. MUG is widely regarded as a “single turnover” enzyme because it still remains tightly bound to its abasic product after cleavage, thus impeding its catalytic turnover. This may be a general protective mechanism to protect the abasic BER intermediate, whereby coordination of enzyme activity in BER is achieved through displacement of the DNA glycosylase by the downstream apurinic-apyrimidinic (AP) endonuclease. Numerous DNA glycosylases have now been cited as having an enhanced turnover in the presence of an AP endonuclease. The aim of this project is to investigate enzyme coordination between MUG and its both downstream AP endonucleases, Exonuclease III (ExoIII) and Endonuclease IV (EndoIV), in the initial steps of BER. We show here that MUG binds its substrate, abasic DNA and non-specific DNA in the differential modes. A 2:1 cooperative binding stoichiometry with abasic DNA is demonstrated to be of functional significance in both product binding and catalysis via fluorescence anisotropy assays, band shift assays and loss-of-function site-directed mutagenesis methods. The effects of the ExoIII and EndoIV on the MUG turnover kinetics with a U•G containing substrate was investigated. Both ExoIII and EndoIV greatly enhance the turnover of MUG. Furthermore, the analysis of both ExoIII catalytic activity dependent and concentration dependent on MUG turnover demonstrate ExoIII may employ a product scavenging mechanism to enhance MUG turnover. These combined results constitute a new concept that MUG has a pre-catalytic discrimination ability to coordinate its reactivity behavior with the other enzymes.Open Acces

Transcriptional Cis-Regulatory Elements and Trans-Factors of a Gene for the Seed Storage Protein Beta-Phaseolin From Common Bean (Phaseolus Vulgaris).

In many dicotyledons, the nutrient reserves are stored in the cotyledons. Phaseolin, 7S globulin, is the major seed protein from common bean Phaseolus vulgaris. In vitro transcription assays and gene transfer studies indicate that phaseolin gene expression is regulated primarily at the transcriptional level. In this dissertation, I studied cis- and trans-acting factors that may play a role in the transcriptional regulation of the $\beta$-phaseolin gene. Gel mobility shift and exonuclease III protection assays identified four distinct DNA binding proteins, CAN, AG-1, CA-1, and TATA-box binding protein. Three CANNTG motifs, CACGTG ($-$248/$-$243), CACCTG ($-$163/$-$158), and CATATG ($-$100/$-$95), were found to be preferred target sequences of CAN. The cis-activities of CAN and AG-1 binding sites were studied systematically by substitution mutations. The results indicate that the CACGTG (G-box) motif a major positive cis-element and acts synergistically with the CACCTG motif. The results also show that AG-I binding sites function as major positive ($-$191/$-$182) and negative ($-$376/$-$367,$-$356/$-$347) cis-elements. These results led me to hypothesize that CAN and AG-1 play a major role in the transcriptional regulation of the $\beta$-phaseolin gene. As a first step to understand the molecular nature of CAN, a bean seed cDNA library was constructed and screened for proteins capable of binding to oligonucleotides containjng the phaseolin G-box. Three positive clones were identified and further studied. DNA sequencing analyses indicate that the three cDNAs encode two homologous proteins that belong to the basic region/helix-loop-llelix (bHLH) protein family. Gel mobility shift assays with the proteins, PG1 and PG2, expressed in E. coli indicate that the two bHLH proteins preferentially bind to the G-box among the three phaseolin E-boxes. Northern blot analysis showed that PG1 is expressed constitutively in the plant and that PG2 is expressed primarily in the root. Based on these and other results, the transcriptional regulation of the $\beta$-phaseolin gene is discussed in relationship to the embryogenesis of common bean

The Role and Molecular Mechanisms of Wss1 in Preserving Genomic Stability

Cells are constantly under threat from both exogenous and endogenous sources of DNA damage. Eukaryotic organisms, however, possess conserved mechanisms that accurately and faithfully respond to DNA damage. The inability to effectively remove DNA lesions can lead to an accumulation of mutations which can compromise cellular viability. The DNA damage response is conserved from bacteria to eukaryotic organisms and have been well characterized, however, how covalently crosslinked proteins are removed from DNA remains enigmatic This thesis provides genetic and biochemical evidence implicating Wss1, a yeast metalloprotease in genome maintenance. We have identified SUMOylation to be an important signal that mediates the removal of as DNA-protein crosslinks. DNA protein crosslinks (DPC) are lethal lesions which are covalently linked to DNA. These lesions can impeding essential DNA transactions including chromosome duplication, chromatin remodeling and gene transcription. We characterized Siz1 and Mms21 E3 SUMO ligases to be important for modifying DPCs. To further expand the role of Mms21 in DPC repair we probed the impact of structural maintenance of chromosome (SMC) mutants in repairing DPCs. This thesis shows that Wss1 is involved in cleaving histones in order to prevent their accumulation during hydroxyurea induced replication stress. In vitro cleavage assays with purified proteins indicate that Wss1’s histone H3 cleavage activity is dependent on its protease activity alone and not its SUMO binding nor p97 domains, unlike in Wss1- mediated removal of DNA-protein crosslinks. Together, we provide molecular evidence suggesting that Wss1 is an important mediator in genome maintenance

Nucleobase, Nucleoside, And Neighboring Nucleotides: Intrinsic Preferences For Tet Enzyme-Mediated Oxidation Of 5-Methylcytosine

The Ten-eleven-translocation (TET) family of enzymes can oxidize the fifth base of DNA, 5-methylcytosine (mC) sequentially, to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). The biochemical preference of TET enzymes for these substrates, in the canonical cytosine guanine dinucleotides (CpG), mimics the order in which they are generated and is reflected in levels of these oxidized modifications (oxmCs) detected in various genomes. Other than this exception, there is conflicting or limited data concerning intrinsic substrate preferences of TET, particularly with regards to different nucleic acid structures, sequence contexts, and extent to which TET mediates oxmCs in clustered proximity to one another. Thus, in this thesis, I present our efforts to determine intrinsic substrate preferences of TET enzymes, and in doing so expand upon our understanding of mechanisms driving these relative activities and the functional significance of observed levels of oxmCs in vivo. After a review of the field, in Chapter 2, I present our work comparing TET activity on different DNA and RNA structures in vitro. We found that TET is relatively promiscuous on a variety of DNA/RNA structures but prefers DNA, a specificity that is dictated by nucleic acid identity of the target base, as well helical conformation of the substrate. In Chapter 3, I newly expose the relative tolerance of TET activity on hmC with a non-G at the +1 base, although mCpG is still largely preferred. This tolerance for hmC oxidation by TET and fC and caC excision by TDG, regardless of the +1 base, supports a model explaining hmCpH depletion relative to mCpH and hmCpG in some genomes. In Chapter 4, I narrate our efforts to quantify clusters of oxmCs using modification-specific sequencing methods and observe that TET is intrinsically capable of clusters of at least fC and caC. Also, we explore the possibility that these clusters are mediated by either strand processivity of TET or underlying sequence context preferences. Finally, I propose two kinetics-based experiments to test our hypotheses regarding mechanisms driving these substrate preferences, along with ways to exploit our knowledge of TET enzymes for creation of more efficient and specific epigenetic editing tools