Cas12a (Cpf1) is a representative type V-A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) effector RNA-guided DNA endonuclease used widely for genome editing. Identification of Cas12a inhibitors is important for regulating gene editing, enhancing genome editing specificity, and safety for human therapeutics. This study used a fluorescence-based assay to screen diverse drug libraries at a core facility for potential small molecule candidates that can inhibit AsCas12a endonuclease activities. Further validation of the major hit compounds revealed that these small molecules inhibit Cas12a in vitro DNA cis and trans cleavage activities as well as gene editing in cells. IC50 values obtained from gene editing inhibition were even lower than primary screening reported IC50. We determined the impact of the small molecules on the thermal stability of Cas12a, possible binding sites, and binding affinity (Kd) using thermal denaturation experiments. Enzyme kinetics studies were used to investigate the effect of the inhibitors on ribonucleoprotein complex formation. The discovered molecules create a tool for achieving safer applications of CRISPR-Cas12a in biotechnology and therapeutics.

The fidelity of DNA replication prevents mutations that may lead to cancer predisposition or neurodegenerative diseases. One mechanism that enhances DNA replication fidelity is DNA mismatch repair, which corrects mismatches and small insertion/deletion loops that have escaped polymerase proofreading. In all eukaryotes and most prokaryotes, MutL (a key mismatch repair protein) has an intrinsic endonuclease activity that nicks the newly synthesized strand and recruits downstream factors to remove and correct errors. It has been proposed that ATP binding promotes a series of conformational changes that induce structural order within MutL and stimulates its endonuclease activity. The C-terminal domain of MutL, which harbors the endonuclease site, does not bind to DNA. This has prevented the molecular characterization of its endonuclease activity. In this thesis, we first show that MutL in B. subtilis exhibits asymmetric conformations similar to yeast and human MutL homologs. We also devise a novel approach to bypass the binding defect of the C-terminal domain by using fusion proteins. We find that these fusions bind to DNA specifically and, in the presence of the processivity clamp, can nick DNA. One of these fusion proteins in particular stimulates the nicking activity much more efficiently than the C-terminal domain alone. This work lays the foundation for the mechanistic characterization of the MutL endonuclease and provides a method to stabilize transient protein-DNA interactions. / Thesis / Master of Science (MSc)

No description available.

DNA mismatch repair corrects base-base mismatches and small insertion/deletion loops generated during normal DNA replication. If left unrepaired, these errors become permanent mutations and can lead to increased susceptibility to cancer. In most prokaryotes and all eukaryotes, the mismatch repair protein MutL is a sequence-unspecific endonuclease that plays an essential role in the strand discrimination step of this pathway. Prokaryotic MutL forms homodimers with two endonuclease sites, whereas eukaryotic MutL homologs form heterodimers with a single active site. To elucidate the mechanistic differences between prokaryotic and eukaryotic MutL, we tested whether both endonuclease sites are necessary for prokaryotic MutL nicking activity. MutL interaction with the processivity clamp is required to stimulate endonuclease activity. Therefore, we also tested whether both subunits of the MutL dimer needed to interact with the processivity clamp. To this end, we engineered a system to independently manipulate each protomer of the homodimer. We demonstrated that prokaryotic MutL is regulated by the processivity clamp to act in a similar manner to eukaryotic MutL with only one functional site contributing to the endonuclease activity. We also devised a strategy to stabilize the transient interactions between MutL, the β-clamp, and DNA through disulfide bridge crosslinking and heterobifunctional crosslinking. Stabilizing transient protein-protein and protein-DNA interactions will help optimize future structural studies in obtaining the ternary complex for mechanistic insights to the MutL endonuclease activity and regulation imposed by the β-clamp. / Thesis / Master of Science (MSc)

The Saccharomyces cerevisiae Mms4 and Mus81 proteins are required for repairing DNA alkylation damage, but not damage caused by ionizing radiations. Previous studies have demonstrated that Mms4 and Mus81 form a specific complex in vivo, which functions as an endonuclease specific for branched DNA molecules. <p> In an effort to further understand the role of the Mus81-Mms4 complex in vivo, the structural and functional characteristics of this complex were investigated in this study. The epistatic analysis revealed that RAD52 was epistatic to MMS4 with respect to killing by methyl methanesulfonate (MMS), suggesting that MMS4 is involved in the RAD52 dependent homologous recombinational repair pathway. However, the mms4 rad51, mms4 rad54 and mms4 rad50 double mutants showed more sensitivity to MMS than either corresponding single gene disruptant. Since Rad51 and Rad54 are required to form the Holliday junction during recombinational repair pathway, it is unlikely that the Mus81-Mms4 complex functions as a Holliday junction resolvase in vivo. <p> The role of MMS4 in DNA damage induced mutagenesis has been investigated. Deletion of MMS4 had no obvious effects on damage-induced basepair mutations, but increased frame-shift mutations by 3 fold when the yeast cells were treated with MMS. This suggests that the Mus81-Mms4 complex plays a role in limiting the damage-induced frame-shift mutagenesis. <p> Through a yeast two-hybrid assay, Mus81 and Mms4 have been demonstrated to form a stable and specific complex in vivo. This result is consistent with previous studies. To localize the domains of the Mms4 and Mus81 proteins involved in herterodimer formation, a series of deletion mutants were constructed for the yeast two-hybrid assay. The Mus81-binding domain of Mms4 was mapped to the extreme C-terminal region between amino acids 598-691. The Mms4-binding domain of Mus81 was mapped to a domain between amino acids 527-632. The results from co-immunoprecipitation experiment were consistent with those from the yeast two-hybrid assay. The Mms4-1 (Gly173Arg) protein was found to lose its interaction with Mus81, and this kind of amino acid substitution is very likely to alter the three-dimension structure of the protein. Thus we hypothesize that the three-dimensional structure is also important for Mms4 to interact with Mus81. <p> By studies on green fluorescent protein (GFP) fusion proteins and their subcellular localization, we demonstrated that Mms4 and Mus81 are nuclear proteins. When the putative nuclear localization sequence 1 (residues 244-263) in Mms4 was deleted, the truncated protein lost the ability to enter the nucleus. On the contrary, deletion of the putative nuclear localization sequence 2 (residues 539-555) had no effect on the localization of the protein. Furthermore, the nuclear localization of Mus81 was proven to be independent of its interaction with Mms4, and the N-terminal half of Mus81 is necessary and sufficient for its localization to the nucleus.

The Saccharomyces cerevisiae Mms4 and Mus81 proteins are required for repairing DNA alkylation damage, but not damage caused by ionizing radiations. Previous studies have demonstrated that Mms4 and Mus81 form a specific complex in vivo, which functions as an endonuclease specific for branched DNA molecules. <p> In an effort to further understand the role of the Mus81-Mms4 complex in vivo, the structural and functional characteristics of this complex were investigated in this study. The epistatic analysis revealed that RAD52 was epistatic to MMS4 with respect to killing by methyl methanesulfonate (MMS), suggesting that MMS4 is involved in the RAD52 dependent homologous recombinational repair pathway. However, the mms4 rad51, mms4 rad54 and mms4 rad50 double mutants showed more sensitivity to MMS than either corresponding single gene disruptant. Since Rad51 and Rad54 are required to form the Holliday junction during recombinational repair pathway, it is unlikely that the Mus81-Mms4 complex functions as a Holliday junction resolvase in vivo. <p> The role of MMS4 in DNA damage induced mutagenesis has been investigated. Deletion of MMS4 had no obvious effects on damage-induced basepair mutations, but increased frame-shift mutations by 3 fold when the yeast cells were treated with MMS. This suggests that the Mus81-Mms4 complex plays a role in limiting the damage-induced frame-shift mutagenesis. <p> Through a yeast two-hybrid assay, Mus81 and Mms4 have been demonstrated to form a stable and specific complex in vivo. This result is consistent with previous studies. To localize the domains of the Mms4 and Mus81 proteins involved in herterodimer formation, a series of deletion mutants were constructed for the yeast two-hybrid assay. The Mus81-binding domain of Mms4 was mapped to the extreme C-terminal region between amino acids 598-691. The Mms4-binding domain of Mus81 was mapped to a domain between amino acids 527-632. The results from co-immunoprecipitation experiment were consistent with those from the yeast two-hybrid assay. The Mms4-1 (Gly173Arg) protein was found to lose its interaction with Mus81, and this kind of amino acid substitution is very likely to alter the three-dimension structure of the protein. Thus we hypothesize that the three-dimensional structure is also important for Mms4 to interact with Mus81. <p> By studies on green fluorescent protein (GFP) fusion proteins and their subcellular localization, we demonstrated that Mms4 and Mus81 are nuclear proteins. When the putative nuclear localization sequence 1 (residues 244-263) in Mms4 was deleted, the truncated protein lost the ability to enter the nucleus. On the contrary, deletion of the putative nuclear localization sequence 2 (residues 539-555) had no effect on the localization of the protein. Furthermore, the nuclear localization of Mus81 was proven to be independent of its interaction with Mms4, and the N-terminal half of Mus81 is necessary and sufficient for its localization to the nucleus.

­^¤åºK­n Multiplex PCR amplification of 16S rRNA gene¡BvirA¡Btpl¡Band H1d genes was developed enabling simultaneous detection in Escherichia coli¡Aan indicator of fecal contamination and food-borne microbial pathogens¡AShigella flexneri¡BCitrobacter freundii¡BSalmonella typhi¡BVibrio cholerae¡BVibrio parahaemolyticus¡Band Staphylococcus aureus¡CEach of the nine pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted gene¡CThe optimized multiplex PCR reaction utilized a primer annealing temperature of 59 ¢Jand used agarose gel electrophoresis for detection of the PCR-amplified products¡CSelection of appropriate target genes¡Boligonucleotide primers ¡BPCR reaction¡Band cycling parameters resulted in the amplification of four target genes simultaneously in a single PCR reaction with the sensitivity of detection was 102 CFU after 32 cycles¡CMultiplex PCR amplification followed by differential PCR for E. coli / Shigella¡A and Citrobacter / Salmonella¡Asequenced for the PCR-amplified products of 16S rRNA gene of the seven pathogens in this study¡Aand used restriction endonuclease AfaI to confirm the PCR-amplified products of V. cholerae¡AV. parahaemolyticus and Staphylococcus aureus¡Ahas been shown to be an sensitive¡Aspecific¡Aand rapid method to detect food-borne bacterial pathogens¡C

Intragenic regions (introns) are found in all classes of organism. Transcription of such genes must undergo a splicing reaction to produce the mature, functional form of RNAs. Introns can be divided into four categories by their splicing mechanisms, namely Group I, Group II, spliceosomal, and nuclear tRNA introns. The former two are self-splicing introns. Group I introns are ubiquitous, however, most metazoan mitochondrial genomes lack introns. A novel group I intron in the mitochondrial cytochrome oxidase I gene (cox1) of Cinachyrella auctraliensis, which belongs to the IB2 subgroup, encodes a putative homing endonuclease with two amino acid motifs of the LAGLIDADG family. The homing endonuclease may perform intron translocation. Splicing in the cox1 of the sponge was demonstrated by comparing the length of DNA and RNA sequences. The intron was spliced in vivo or in vitro as revealed by RT-PCR and sequencing. Group I introns are classified as ribozymes. The pre-mRNAs fold into specific configurations that facilitate attacks of free guanosine followed by two consecutive trans-esterification steps to remove the introns. The excised cox1 intron was found to form a circle with the 5¡¦-end linked to the 3¡¦-end. Two other forms of lariats were also found with the 5¡¦-end linked to the inside sequence of the intron. Mutagenesis of a key nucleotide, which participates base pairing of RNA secondary structure, in P7 region decreased the splicing activity of the intron.

Bacteriophages are viruses that infect bacteria. This thesis describes studies of gp74 from the bacteriophage HK97, which functions as an HNH endonuclease. HNH endonucleases are DNA digestion proteins characterized by two highly conserved His residues and an Asn residue. Like other HNH endonucleases, the activity of gp74 is dependent on binding of divalent metal ions to the HNH motif. Current work focused on confirming the identity of conserved HNH motif residues of gp74. We hypothesized the catalytic His residue is H43, the structural Asn residue is N73, and that H82 is involved in metal–binding. Additional residues in the ββα–fold, such as D42, may also bind the metal. Our bound metal analysis and the sequence of gp74 also suggest the presence of a Zn2+–finger motif. Mutations of D42 and H82 decrease the activity of gp74, without affecting the structure. These studies advance our understanding of the gp74 activity.

Bacteriophages are viruses that infect bacteria. This thesis describes studies of gp74 from the bacteriophage HK97, which functions as an HNH endonuclease. HNH endonucleases are DNA digestion proteins characterized by two highly conserved His residues and an Asn residue. Like other HNH endonucleases, the activity of gp74 is dependent on binding of divalent metal ions to the HNH motif. Current work focused on confirming the identity of conserved HNH motif residues of gp74. We hypothesized the catalytic His residue is H43, the structural Asn residue is N73, and that H82 is involved in metal–binding. Additional residues in the ββα–fold, such as D42, may also bind the metal. Our bound metal analysis and the sequence of gp74 also suggest the presence of a Zn2+–finger motif. Mutations of D42 and H82 decrease the activity of gp74, without affecting the structure. These studies advance our understanding of the gp74 activity.