Bruton’s tyrosine kinase (Btk) is a non-receptor tyrosine kinase required for regulation of B-lym... more Bruton’s tyrosine kinase (Btk) is a non-receptor tyrosine kinase required for regulation of B-lymphocyte development, differentiation and signalling. Deregulation of Btk signalling leads to cancer and immunodeficiency diseases like X-linked agammaglobulinemia (XLA) for human and X-linked immunodeficiency (Xid) for mice. Btk is composed of an N-terminal Pleckstrin and Tec homology domain (PH-TH), Src homology 2 and 3 (SH₂ and SH₃) domains and the C-terminal tyrosine kinase domain. Btk is known to be activated by its localization to the plasma membrane and subsequent phosphorylation by Src family of kinases. Recently, it was found that inositol hexakisphosphate (IP₆) activates Btk by binding to the PH-TH domain, even in the absence of a membrane. This may represent an alternate mechanism of activation of Btk. The detailed structural and functional significance of Btk activation by IP₆ is still unknown. The E41K substitution mutation in the PH-TH domain constitutively activates Btk. This activating mutant also shows higher affinity for IP₆ than the wild type Btk emphasising the central role of IP₆ in Btk regulation. In this study, we will focus our efforts to understand the role of IP₆ in regulation of Btk and it’s constitutively activated mutant E41K using Dynamic Light Scattering and Size Exclusion Chromatography.
The human MCM8-9 helicase functions in concert with HROB in the context of homologous recombinati... more The human MCM8-9 helicase functions in concert with HROB in the context of homologous recombination, but its precise function is unknown. To gain insights into how HROB regulates MCM8-9, we first used molecular modeling and biochemistry to define their interaction interface. We show that HROB makes important contacts with both MCM8 and MCM9 subunits, which directly promotes its DNA-dependent ATPase and helicase activities. MCM8-9-HROB preferentially binds and unwinds branched DNA structures, and single-molecule experiments reveal a low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexameric complex that assembles from dimers on DNA in the presence of ATP, which is prerequisite for its helicase function. The hexamer formation thus involves two repeating protein-protein interfaces forming between the alternating MCM8 and MCM9 subunits. One of these interfaces is rather stable and forms an obligate heterodimer, while the other interface is labile and mediates the assembly of the ...
Microbial Contamination and Food Degradation, 2018
Abstract Microbial association with dry and fresh food ingredients has impacted the culture, econ... more Abstract Microbial association with dry and fresh food ingredients has impacted the culture, economy, and health of our society in a positive, as well as in a negative way for centuries. Fruits, vegetables, dairy products, and even whole grain and cereals are subjected intentionally to microbial praxis for furtherance of food commodity. Negative influences of bacteria, yeasts, moulds, and viruses predominantly cause enteric diseases by intoxicating meat, poultry, and milk products with microbial enzymes and toxins. Intrinsic factors, such as nutrient content, pH, redox potential, and water activity in association with extrinsic factors, such as temperature, relative humidity, and gaseous atmosphere, induce unintentional microbial contamination in food supplements, resulting in quality deterioration. Moisture content also determines the category of food spoilage. Rancidification of lipid bodies and proteolysis of amino contents enhances the chemical degradation of food materials with time. Additional unhygienic practices transmit contamination factor during food processing and packaging which demands a stringent quality control. To implement such quality measures, tests, such as microscopic methods, flow cytometry, LAL test for endotoxin, PCR and so on, can be performed.This chapter explores the diversity of microbes and the underlying processes associated with food degradation and the concomitant health issues, with special emphasis on certain ailments, such as Cancer, Hepatitis, Diarrhea, and Fever. It also intends to discuss the techniques to scrutinize and check food quality with potential ways to implement it.
SummaryDuring prophase of the first meiotic division, cells deliberately break their DNA. These D... more SummaryDuring prophase of the first meiotic division, cells deliberately break their DNA. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables reciprocal exchange of DNA segments between homologous chromosomes, thus promoting genetic diversity in the progeny1. A successful completion of meiotic recombination requires nucleolytic processing of recombination intermediates. Genetic and cellular data implicated a pathway dependent on the putative MLH1-MLH3 (MutLγ) nuclease in generating crossovers, but mechanisms that lead to its activation were unclear2–4. Here, we have biochemically reconstituted key elements of this pro-crossover pathway. First, we show that human MSH4-MSH5 (MutSγ), which was known to support crossing over5–7, binds branched recombination intermediates and physically associates with MutLγ. This helps stabilize the ensemble at joint molecule structures and adjacent dsDNA. Second, we show that MutSγ dire...
Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maint... more Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. HR is carried out by a complex network of DNA repair factors. Here we identify C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a novel DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we identify a direct interaction with MCM8-9, a putative helicase complex mutated in Primary Ovarian Insufficiency, that is crucial for MCM8IP ability to promote resistance to DNA damaging agents. In addition to its association with MCM8-9, MCM8IP also binds directly to RPA1. We show that the interactions of MCM8IP with both MCM8-9 and RPA are required to maintain replication fork prog...
The Dna2 helicase-nuclease functions in concert with the replication protein A (RPA) in DNA doubl... more The Dna2 helicase-nuclease functions in concert with the replication protein A (RPA) in DNA double-strand break repair. Using ensemble and single-molecule biochemistry, coupled with structure modeling, we demonstrate that the stimulation of S. cerevisiae Dna2 by RPA is not a simple consequence of Dna2 recruitment to single-stranded DNA. The large RPA subunit Rfa1 alone can promote the Dna2 nuclease activity, and we identified mutations in a helix embedded in the N-terminal domain of Rfa1 that specifically disrupt this capacity. The same RPA mutant is instead fully functional to recruit Dna2 and promote its helicase activity. Furthermore, we found residues located on the outside of the central DNA-binding OB-fold domain Rfa1-A, which are required to promote the Dna2 motor activity. Our experiments thus unexpectedly demonstrate that different domains of Rfa1 regulate Dna2 recruitment, and its nuclease and helicase activities. Consequently, the identified separation-of-function RPA var...
Bruton’s tyrosine kinase (Btk) is a non-receptor tyrosine kinase required for regulation of B-lym... more Bruton’s tyrosine kinase (Btk) is a non-receptor tyrosine kinase required for regulation of B-lymphocyte development, differentiation and signalling. Deregulation of Btk signalling leads to cancer and immunodeficiency diseases like X-linked agammaglobulinemia (XLA) for human and X-linked immunodeficiency (Xid) for mice. Btk is composed of an N-terminal Pleckstrin and Tec homology domain (PH-TH), Src homology 2 and 3 (SH₂ and SH₃) domains and the C-terminal tyrosine kinase domain. Btk is known to be activated by its localization to the plasma membrane and subsequent phosphorylation by Src family of kinases. Recently, it was found that inositol hexakisphosphate (IP₆) activates Btk by binding to the PH-TH domain, even in the absence of a membrane. This may represent an alternate mechanism of activation of Btk. The detailed structural and functional significance of Btk activation by IP₆ is still unknown. The E41K substitution mutation in the PH-TH domain constitutively activates Btk. Th...
Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maint... more Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. Here we characterize C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we report that MCM8IP directly associates with MCM8-9, a helicase complex mutated in primary ovarian insufficiency, and RPA1. We additionally show that the interactions of MCM8IP with MCM8-9 and RPA facilitate HR and promote replication fork progression and cellular viability in response to treatment with crosslinking agents. Mechanistically, MCM8IP stimulates the helicase activity of MCM8-9. Collectively, our work identifies MCM8IP as a key regula...
Bruton’s tyrosine kinase (Btk) is a non-receptor tyrosine kinase required for regulation of B-lym... more Bruton’s tyrosine kinase (Btk) is a non-receptor tyrosine kinase required for regulation of B-lymphocyte development, differentiation and signalling. Deregulation of Btk signalling leads to cancer and immunodeficiency diseases like X-linked agammaglobulinemia (XLA) for human and X-linked immunodeficiency (Xid) for mice. Btk is composed of an N-terminal Pleckstrin and Tec homology domain (PH-TH), Src homology 2 and 3 (SH₂ and SH₃) domains and the C-terminal tyrosine kinase domain. Btk is known to be activated by its localization to the plasma membrane and subsequent phosphorylation by Src family of kinases. Recently, it was found that inositol hexakisphosphate (IP₆) activates Btk by binding to the PH-TH domain, even in the absence of a membrane. This may represent an alternate mechanism of activation of Btk. The detailed structural and functional significance of Btk activation by IP₆ is still unknown. The E41K substitution mutation in the PH-TH domain constitutively activates Btk. This activating mutant also shows higher affinity for IP₆ than the wild type Btk emphasising the central role of IP₆ in Btk regulation. In this study, we will focus our efforts to understand the role of IP₆ in regulation of Btk and it’s constitutively activated mutant E41K using Dynamic Light Scattering and Size Exclusion Chromatography.
The human MCM8-9 helicase functions in concert with HROB in the context of homologous recombinati... more The human MCM8-9 helicase functions in concert with HROB in the context of homologous recombination, but its precise function is unknown. To gain insights into how HROB regulates MCM8-9, we first used molecular modeling and biochemistry to define their interaction interface. We show that HROB makes important contacts with both MCM8 and MCM9 subunits, which directly promotes its DNA-dependent ATPase and helicase activities. MCM8-9-HROB preferentially binds and unwinds branched DNA structures, and single-molecule experiments reveal a low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexameric complex that assembles from dimers on DNA in the presence of ATP, which is prerequisite for its helicase function. The hexamer formation thus involves two repeating protein-protein interfaces forming between the alternating MCM8 and MCM9 subunits. One of these interfaces is rather stable and forms an obligate heterodimer, while the other interface is labile and mediates the assembly of the ...
Microbial Contamination and Food Degradation, 2018
Abstract Microbial association with dry and fresh food ingredients has impacted the culture, econ... more Abstract Microbial association with dry and fresh food ingredients has impacted the culture, economy, and health of our society in a positive, as well as in a negative way for centuries. Fruits, vegetables, dairy products, and even whole grain and cereals are subjected intentionally to microbial praxis for furtherance of food commodity. Negative influences of bacteria, yeasts, moulds, and viruses predominantly cause enteric diseases by intoxicating meat, poultry, and milk products with microbial enzymes and toxins. Intrinsic factors, such as nutrient content, pH, redox potential, and water activity in association with extrinsic factors, such as temperature, relative humidity, and gaseous atmosphere, induce unintentional microbial contamination in food supplements, resulting in quality deterioration. Moisture content also determines the category of food spoilage. Rancidification of lipid bodies and proteolysis of amino contents enhances the chemical degradation of food materials with time. Additional unhygienic practices transmit contamination factor during food processing and packaging which demands a stringent quality control. To implement such quality measures, tests, such as microscopic methods, flow cytometry, LAL test for endotoxin, PCR and so on, can be performed.This chapter explores the diversity of microbes and the underlying processes associated with food degradation and the concomitant health issues, with special emphasis on certain ailments, such as Cancer, Hepatitis, Diarrhea, and Fever. It also intends to discuss the techniques to scrutinize and check food quality with potential ways to implement it.
SummaryDuring prophase of the first meiotic division, cells deliberately break their DNA. These D... more SummaryDuring prophase of the first meiotic division, cells deliberately break their DNA. These DNA breaks are repaired by homologous recombination, which facilitates proper chromosome segregation and enables reciprocal exchange of DNA segments between homologous chromosomes, thus promoting genetic diversity in the progeny1. A successful completion of meiotic recombination requires nucleolytic processing of recombination intermediates. Genetic and cellular data implicated a pathway dependent on the putative MLH1-MLH3 (MutLγ) nuclease in generating crossovers, but mechanisms that lead to its activation were unclear2–4. Here, we have biochemically reconstituted key elements of this pro-crossover pathway. First, we show that human MSH4-MSH5 (MutSγ), which was known to support crossing over5–7, binds branched recombination intermediates and physically associates with MutLγ. This helps stabilize the ensemble at joint molecule structures and adjacent dsDNA. Second, we show that MutSγ dire...
Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maint... more Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. HR is carried out by a complex network of DNA repair factors. Here we identify C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a novel DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we identify a direct interaction with MCM8-9, a putative helicase complex mutated in Primary Ovarian Insufficiency, that is crucial for MCM8IP ability to promote resistance to DNA damaging agents. In addition to its association with MCM8-9, MCM8IP also binds directly to RPA1. We show that the interactions of MCM8IP with both MCM8-9 and RPA are required to maintain replication fork prog...
The Dna2 helicase-nuclease functions in concert with the replication protein A (RPA) in DNA doubl... more The Dna2 helicase-nuclease functions in concert with the replication protein A (RPA) in DNA double-strand break repair. Using ensemble and single-molecule biochemistry, coupled with structure modeling, we demonstrate that the stimulation of S. cerevisiae Dna2 by RPA is not a simple consequence of Dna2 recruitment to single-stranded DNA. The large RPA subunit Rfa1 alone can promote the Dna2 nuclease activity, and we identified mutations in a helix embedded in the N-terminal domain of Rfa1 that specifically disrupt this capacity. The same RPA mutant is instead fully functional to recruit Dna2 and promote its helicase activity. Furthermore, we found residues located on the outside of the central DNA-binding OB-fold domain Rfa1-A, which are required to promote the Dna2 motor activity. Our experiments thus unexpectedly demonstrate that different domains of Rfa1 regulate Dna2 recruitment, and its nuclease and helicase activities. Consequently, the identified separation-of-function RPA var...
Bruton’s tyrosine kinase (Btk) is a non-receptor tyrosine kinase required for regulation of B-lym... more Bruton’s tyrosine kinase (Btk) is a non-receptor tyrosine kinase required for regulation of B-lymphocyte development, differentiation and signalling. Deregulation of Btk signalling leads to cancer and immunodeficiency diseases like X-linked agammaglobulinemia (XLA) for human and X-linked immunodeficiency (Xid) for mice. Btk is composed of an N-terminal Pleckstrin and Tec homology domain (PH-TH), Src homology 2 and 3 (SH₂ and SH₃) domains and the C-terminal tyrosine kinase domain. Btk is known to be activated by its localization to the plasma membrane and subsequent phosphorylation by Src family of kinases. Recently, it was found that inositol hexakisphosphate (IP₆) activates Btk by binding to the PH-TH domain, even in the absence of a membrane. This may represent an alternate mechanism of activation of Btk. The detailed structural and functional significance of Btk activation by IP₆ is still unknown. The E41K substitution mutation in the PH-TH domain constitutively activates Btk. Th...
Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maint... more Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. Here we characterize C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we report that MCM8IP directly associates with MCM8-9, a helicase complex mutated in primary ovarian insufficiency, and RPA1. We additionally show that the interactions of MCM8IP with MCM8-9 and RPA facilitate HR and promote replication fork progression and cellular viability in response to treatment with crosslinking agents. Mechanistically, MCM8IP stimulates the helicase activity of MCM8-9. Collectively, our work identifies MCM8IP as a key regula...
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