DNA post-replication repair (PRR) is a cellular tolerance mechanism by which eukaryotic cells survive lethal lesions during or after DNA synthesis. In the yeast Saccharomyces cerevisiae, modification of proliferating cell nuclear antigen (PCNA) by ubiquitin and by small ubiquitin-like modifier (SUMO) plays an important role in PRR. PCNA ubiquitination is dependent on Rad6, a ubiquitin-conjugating enzyme (E2) and Rad18, a ubiquitin ligase (E3). Rad6 and Rad18 form a stable complex. PCNA sumoylation is dependent on Ubc9, an E2 specific to SUMO modification. <p>PRR in mammalian cells is less well understood. However, human Rad18 (hRad18) has been found to interact with human Rad6 (HHR6A/B). In this study, we detected physical interaction between hRad18 and human Ubc9 (hUbc9) through yeast two-hybrid assays. In order to define the domain(s) of hRad18 involved in the formation of a complex with HHR6B or hUbc9, a series of yeast two-hybrid constructs containing various hRAD18 gene deletions and mutations were made. A C-terminal region of hRad18, containing the putative HHR6A/B binding domain (amino acids 340 to 395), interacts with HHR6A/B while the N-terminus (amino acids 1-93) does not. Yeast Rad18 has a homologous fragment of the HHR6A/B binding domain and this fragment is sufficient to interact with yeast Rad6 in yeast two-hybrid assays, so we infer that hRad18 interacts with HHR6B through the same domain. Surprisingly, both the N-terminal and C-terminal fragments of hRad18 can interact with hUbc9, suggesting the existence of two separate domains in hRad18 interacting with hUbc9. The N-terminal fragment of hRad18 contains only a RING finger domain (amino acids 25-64), which is probably responsible for binding to hUbc9. The C-terminal fragment of hRad18 with HHR6A/B binding domain deletion can still interact with hUbc9, suggesting that the HHR6A/B binding domain is not involved in hUbc9 interaction. A key cysteine mutation (C28F) in the RING finger domain abolished the interactions of hRad18 with both HHR6A/B and hUbc9. This amino acid substitution is likely to alter the three-dimensional structure of the protein, thus making the protein unstable. Taken together, results obtained from this study suggest that hRad18 may regulate the modification status of PCNA by interacting with two different E2s, HHR6A/B and hUbc9, through distinct domains.

DNA post-replication repair (PRR) is a cellular tolerance mechanism by which eukaryotic cells survive lethal lesions during or after DNA synthesis. In the yeast Saccharomyces cerevisiae, modification of proliferating cell nuclear antigen (PCNA) by ubiquitin and by small ubiquitin-like modifier (SUMO) plays an important role in PRR. PCNA ubiquitination is dependent on Rad6, a ubiquitin-conjugating enzyme (E2) and Rad18, a ubiquitin ligase (E3). Rad6 and Rad18 form a stable complex. PCNA sumoylation is dependent on Ubc9, an E2 specific to SUMO modification. <p>PRR in mammalian cells is less well understood. However, human Rad18 (hRad18) has been found to interact with human Rad6 (HHR6A/B). In this study, we detected physical interaction between hRad18 and human Ubc9 (hUbc9) through yeast two-hybrid assays. In order to define the domain(s) of hRad18 involved in the formation of a complex with HHR6B or hUbc9, a series of yeast two-hybrid constructs containing various hRAD18 gene deletions and mutations were made. A C-terminal region of hRad18, containing the putative HHR6A/B binding domain (amino acids 340 to 395), interacts with HHR6A/B while the N-terminus (amino acids 1-93) does not. Yeast Rad18 has a homologous fragment of the HHR6A/B binding domain and this fragment is sufficient to interact with yeast Rad6 in yeast two-hybrid assays, so we infer that hRad18 interacts with HHR6B through the same domain. Surprisingly, both the N-terminal and C-terminal fragments of hRad18 can interact with hUbc9, suggesting the existence of two separate domains in hRad18 interacting with hUbc9. The N-terminal fragment of hRad18 contains only a RING finger domain (amino acids 25-64), which is probably responsible for binding to hUbc9. The C-terminal fragment of hRad18 with HHR6A/B binding domain deletion can still interact with hUbc9, suggesting that the HHR6A/B binding domain is not involved in hUbc9 interaction. A key cysteine mutation (C28F) in the RING finger domain abolished the interactions of hRad18 with both HHR6A/B and hUbc9. This amino acid substitution is likely to alter the three-dimensional structure of the protein, thus making the protein unstable. Taken together, results obtained from this study suggest that hRad18 may regulate the modification status of PCNA by interacting with two different E2s, HHR6A/B and hUbc9, through distinct domains.

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.

No description available.

The type II secretion system is used by many pathogenic and non-pathogenic bacteria for the extracellular secretion of enzymes and toxins. <i>Aeromonas hydrophila</i> is a Gram-negative pathogen that secretes proteins via the type II secretion system.<p> In the studies described here, a series of yeast two-hybrid assays was performed to identify protein-protein interactions in the type II secretion system of <i>A. hydrophila</i>. The periplasmic domains of ExeA and ExeB were assayed for interactions with the periplasmic domains of Exe A, B, C, D, K, L, M, and N. Interactions were observed for both ExeA and ExeB with the secretin ExeD in one orientation. In addition, a previously identified interaction between ExeC and ExeD was observed. In order to further examine and map these interactions, a series of eight two-codon insertion mutations in the amino terminal domain of ExeD was screened against the periplasmic domains of ExeA and ExeB. As a result, the interactions were verified and mapped to subdomains of the ExeD periplasmic domain. To positively identify the region of ExeD involved in the interactions with ExeA, B, C and D, deletion mutants of ExeD were constructed based on the two-codon insertion mutation mapping of subdomains of the ExeD periplasmic domain, and yeast two-hybrid assays were carried out. The results showed that a fragment of the periplasmic domain of ExeD, from amino acid residue 26 to 200 of ExeD, was involved in the interactions with ExeA, B and C. As an independent assay for interactions between ExeAB and the secretin, His-tagged derivatives of the periplasmic domains of ExeA and ExeB were constructed and co-purification on Ni-NTA agarose columns was used to test for interactions with untagged ExeD. These experiments confirmed the interaction between ExeA and ExeD, although there was background in the co-purification test.<p> These results provide support for the hypothesis that the ExeAB complex functions to organize the assembly of the secretin through interactions between both peptidoglycan and the secretin that result in its multimerization into the peptidoglycan and outer membrane layers of the envelope.

Reagents that bind proteins are applicable in biology for detection of molecules, perturbation of signaling pathways and development of small-molecule pharmaceuticals. Protein ligands interact with proteins, inhibiting or altering their function. They are isolated from combinatorial libraries to interact with a specific target, using selection techniques such as phage display or yeast-two-hybrid assay. For the latter, one inconvenience is the detection of false positives, which can be solved by screening pools containing the samples to be tested, instead of individual samples. Samples are distributed in the pools following a pooling design. The PI-deconvolution pooling design was developed to screen cDNA libraries using the yeast-two-hybrid assay, which are smaller in size than protein ligand combinatorial libraries. Modifications to the PI-deconvolution screening technique were developed to adapt it to the screening of protein ligand combinatorial libraries using the yeast-two-hybrid assay. Every spot of the array containing the combinatorial library was randomly pooled. However, the yeast-two-hybrid assay loses sensitivity when strains are pooled. As PI-deconvolution requires detecting every interaction, we determined the optimal amount of library members that can be pooled in a spot, and the optimal number of replicates to ensure the detection of an interaction.<p> The yeast-two-hybrid assay was used to perform a screening of a combinatorial library with seven domains of BCR-ABL, which were pooled according to PI-deconvolution. BCR-ABL is a chimeric protein with unregulated kinase activity that is responsible for chronic myelogenous leukemia. The scaffold used in the combinatorial library was an engineered intein that forms lariat peptides. After a screening of this library was performed, positive interactions were detected in 775 spots of the arrays that contained 1432 positive hits. Only 53 spots were deconvoluted. The coding sequences of the lariat peptides were determined for 23 lariat peptides interacted with the GEF domain of BCR, and for ABL, two with the FABD domain, one with the SH1 domain, and one with the SH3 domain. Finally, a &beta;-galactosidase assay was performed to assess the affinity of the lariat peptides for their target.<p> The isolated lariat peptides are potential inhibitors of BCR-ABL that can have therapeutic potential. This study will improve other screenings of combinatorial libraries with the yeast-two-hybrid assay.

The type II secretion system is used by many pathogenic and non-pathogenic bacteria for the extracellular secretion of enzymes and toxins. <i>Aeromonas hydrophila</i> is a Gram-negative pathogen that secretes proteins via the type II secretion system.<p> In the studies described here, a series of yeast two-hybrid assays was performed to identify protein-protein interactions in the type II secretion system of <i>A. hydrophila</i>. The periplasmic domains of ExeA and ExeB were assayed for interactions with the periplasmic domains of Exe A, B, C, D, K, L, M, and N. Interactions were observed for both ExeA and ExeB with the secretin ExeD in one orientation. In addition, a previously identified interaction between ExeC and ExeD was observed. In order to further examine and map these interactions, a series of eight two-codon insertion mutations in the amino terminal domain of ExeD was screened against the periplasmic domains of ExeA and ExeB. As a result, the interactions were verified and mapped to subdomains of the ExeD periplasmic domain. To positively identify the region of ExeD involved in the interactions with ExeA, B, C and D, deletion mutants of ExeD were constructed based on the two-codon insertion mutation mapping of subdomains of the ExeD periplasmic domain, and yeast two-hybrid assays were carried out. The results showed that a fragment of the periplasmic domain of ExeD, from amino acid residue 26 to 200 of ExeD, was involved in the interactions with ExeA, B and C. As an independent assay for interactions between ExeAB and the secretin, His-tagged derivatives of the periplasmic domains of ExeA and ExeB were constructed and co-purification on Ni-NTA agarose columns was used to test for interactions with untagged ExeD. These experiments confirmed the interaction between ExeA and ExeD, although there was background in the co-purification test.<p> These results provide support for the hypothesis that the ExeAB complex functions to organize the assembly of the secretin through interactions between both peptidoglycan and the secretin that result in its multimerization into the peptidoglycan and outer membrane layers of the envelope.

Reagents that bind proteins are applicable in biology for detection of molecules, perturbation of signaling pathways and development of small-molecule pharmaceuticals. Protein ligands interact with proteins, inhibiting or altering their function. They are isolated from combinatorial libraries to interact with a specific target, using selection techniques such as phage display or yeast-two-hybrid assay. For the latter, one inconvenience is the detection of false positives, which can be solved by screening pools containing the samples to be tested, instead of individual samples. Samples are distributed in the pools following a pooling design. The PI-deconvolution pooling design was developed to screen cDNA libraries using the yeast-two-hybrid assay, which are smaller in size than protein ligand combinatorial libraries. Modifications to the PI-deconvolution screening technique were developed to adapt it to the screening of protein ligand combinatorial libraries using the yeast-two-hybrid assay. Every spot of the array containing the combinatorial library was randomly pooled. However, the yeast-two-hybrid assay loses sensitivity when strains are pooled. As PI-deconvolution requires detecting every interaction, we determined the optimal amount of library members that can be pooled in a spot, and the optimal number of replicates to ensure the detection of an interaction.<p> The yeast-two-hybrid assay was used to perform a screening of a combinatorial library with seven domains of BCR-ABL, which were pooled according to PI-deconvolution. BCR-ABL is a chimeric protein with unregulated kinase activity that is responsible for chronic myelogenous leukemia. The scaffold used in the combinatorial library was an engineered intein that forms lariat peptides. After a screening of this library was performed, positive interactions were detected in 775 spots of the arrays that contained 1432 positive hits. Only 53 spots were deconvoluted. The coding sequences of the lariat peptides were determined for 23 lariat peptides interacted with the GEF domain of BCR, and for ABL, two with the FABD domain, one with the SH1 domain, and one with the SH3 domain. Finally, a &beta;-galactosidase assay was performed to assess the affinity of the lariat peptides for their target.<p> The isolated lariat peptides are potential inhibitors of BCR-ABL that can have therapeutic potential. This study will improve other screenings of combinatorial libraries with the yeast-two-hybrid assay.

Ubiquitylation is an indispensable post-translational modification system in eukaryotic cells that leads to the covalent attachment of a small ubiquitin (Ub) protein onto a target. The traditional and best-characterized role for ubiquitylation is a fundamental regulatory mechanism whereby target proteins are tagged with a characteristic Lys48-linked Ub chain that signals for their elimination through proteasomal degradation. Challenging this conventional wisdom is the finding that some ubiquitylated proteins are modified by Ub chains linked through Lys63, providing a molecular signal that is thought to be structurally and functionally distinct from Lys48-linked Ub chains. Of further interest and significance is that the Lys63-linked Ub chains are apparently synthesized through a novel biochemical mechanism employing a unique complex formed between a true Ub conjugating enzyme (E2), Ubc13, and an E2-variant (Uev), Mms2 (or Uev1A). The goal of this thesis was to employ structural and functional approaches in order to better characterize the Ubc13-Mms2 Lys63-linked Ub conjugation complex. <p>Error-free DNA damage tolerance (DDT) in the budding yeast is dependent on Lys63-linked Ub chains synthesized by Ubc13-Mms2 and thus provided the opportunity to experimentally test the function of the human UBC13 and MMS2 genes in a simple model organism. Human UBC13 and MMS2 were each shown to function in place of their yeast counterparts and in accordance, human Ubc13 was shown to physically interact with yeast Mms2, and vice versa. Two human MMS2 homologs were also tested and it was determined that UEV1A but not UEV1B can function in place of mms2 in yeast DDT. Physical interactions were observed between Ubc13 and Uev1A, but not between Ubc13 and Uev1B, suggesting that Ubc13-Uev complex formation is required for function. <p>In collaboration with a research group at the University of Alberta, crystal structure and NMR data were used to develop a mechanistic model for the conjugation of Lys63-linked Ub chains by the Ubc13-Mms2 heterodimer, whereby the special orientation of two Ub molecules facilitates a specific Ub-Ub linkage via Lys63. In order to help support the in vitro model and to determine how the Ubc13-Mms2 structure relates to biological function, I used a structure-based approach to direct the creation of point mutations within four key regions of the Ubc13-Mms2 heterodimer; the Ubc13 active-site, the Ubc13-E3 (Ub ligating enzyme) interface, the Mms2-Ub interface, and the Ubc13-Mms2 interface. <p>Underscoring the importance of the Ub conjugation by Ubc13-Mms2, a Ubc13-C87S active-site mutation was created that could bind to Mms2 but was unable to function in DDT. Regarding the Ubc13-E3 interface, a single Ubc13-M64A point mutation had a potent effect on disrupting Ubc13 function in DDT, as well as its physical interaction with Rad5, TRAF6, and CHFR. The results suggest that different RING finger E3s use the same Ubc13 surface to sequester the Ub conjugation activity of Ubc13-Mms2. Two human Mms2 mutations at Ser32 and Ile62, which are contained within the Mms2-Ub interface, were found to reduce the ability of Mms2 to bind Ub. When the corresponding yeast mutations are combined, a synergistic loss in DDT function is observed. The relative orientation of Ser32 and Ile62 suggests that the Mms2 and Tsg101 Uev families use different Uev surfaces to physically interact with Ub. A 200 ìM dissociation constant for the wild-type Mms2-Ub interaction was also determined. The systematic mutagenesis and testing of 14 Ubc13-Mms2 interface residues led to mutants with partial or complete disruption of binding and function. Using this data, a model involving the insertion of a specific Mms2-Phe residue into a unique Ubc13 hydrophobic pocket was created to explain the specificity of Mms2 for Ubc13, and not other E2s. In addition, the dissociation constant for the wild-type Ubc13-Mms2 heterodimer was determined to be approximately 50 nM. <p>The structural and functional studies strongly support the notion that Ubc13-Mms2 complex has the unique ability to conjugate Lys63-linked Ub chains. However, several reported instances of Lys63-linked Ub chains in vivo have not yet been attributed to Ubc13 or Mms2. To address the disparity I was able to demonstrate and map a physical interaction between Mms2 and Rsp5, an E3 implicated in Lys63-linked Ub conjugation. Surprisingly, it was found that MMS2 is not responsible for the RSP5-dependent Lys63-linked Ub conjugation of a plasma membrane protein. A possible explanation for the apparent paradox is presented.