{"id":{"repo_id":"westminster","oai_identifier":"oai:westminsterresearch.westminster.ac.uk:90xxw"},"canonical_url":"https://search.dev.ndltd.org/etd/westminster/oai:westminsterresearch.westminster.ac.uk:90xxw","repository":{"repo_id":"westminster","name":"University of Westminster","base_url":"https://westminsterresearch.westminster.ac.uk/oai2"},"display":{"title":"Developing as assay to screen inhibitors for various ATP-dependent ligases","abstract":"DNA ligases (EC.6.5.1.1) are key enzymes that catalyze the formation of phosphodiester bonds at single-stranded or double-stranded breaks between adjacent 5’-PO4 and 3’-OH groups of DNA. These enzymes are essential guardians of genomic integrity and have recently been drawing a lot of attention as novel therapeutic targets in anti-bacterial and anticancer therapies. A novel, non-electrophoretic assay method, based on the strength of interaction of the oligonucleotides with Q-sepharose (a strong anion exchanger), was developed to screen inhibitors of DNA ligases from natural product pools as well as chemical libraries. The binding affinities to Q-sepharose resin of a nicked DNA substrate (created from a 30-mer hairpin oligonucleotide and complementary 32P-labelled 6-mer oligonucleotide) and its sealed, ligated product (36-mer) were determined. Initial optimisation studies were performed with T4 DNA ligase, PBCV-1 DNA ligase and a catalytically active form of human DNA ligase I in the presence of doxorubicin (inhibitor of ATP-dependent ligases). These results when analysed in parallel between the conventional electrophoretic assay and the labelled nick-sealing assay showed that the newly developed assay is a reliable non-electrophoretic method in identifying potent DNA ligase inhibitors. The feasibility of the assay was tested in screening a collection of whole cell mass extracts, obtained from a natural product library from Basidiomycetes, in 96-well format. A novel single DNA ligase was identified, expressed and characterised from Trichomonas vaginalis (TV), a pathogenic protozoan parasite. Protein sequence analysis of TV DNA ligase indicates a strong sequence similarity to DNA ligase I homologues. The activity of recombinant TV DNA ligase I (TVlig) was investigated using protein expressed in E.coli cells. The TVlig gene product is 76 kDa and showed optimal ligation activity on a nicked DNA substrate at pH 7-8 in the presence of 1 mM ATP and (8- 20) mM MgCl2 at 30-38oC. The inhibition of the only DNA ligase present in T. vaginalis might suggest for a rational approach to stop replication and hence propagation of the parasite during infection.","abstract_html":"DNA ligases (EC.6.5.1.1) are key enzymes that catalyze the formation of phosphodiester bonds at single-stranded or double-stranded breaks between adjacent 5’-PO4 and 3’-OH groups of DNA. These enzymes are essential guardians of genomic integrity and have recently been drawing a lot of attention as novel therapeutic targets in anti-bacterial and anticancer therapies. A novel, non-electrophoretic assay method, based on the strength of interaction of the oligonucleotides with Q-sepharose (a strong anion exchanger), was developed to screen inhibitors of DNA ligases from natural product pools as well as chemical libraries. The binding affinities to Q-sepharose resin of a nicked DNA substrate (created from a 30-mer hairpin oligonucleotide and complementary 32P-labelled 6-mer oligonucleotide) and its sealed, ligated product (36-mer) were determined. Initial optimisation studies were performed with T4 DNA ligase, PBCV-1 DNA ligase and a catalytically active form of human DNA ligase I in the presence of doxorubicin (inhibitor of ATP-dependent ligases). These results when analysed in parallel between the conventional electrophoretic assay and the labelled nick-sealing assay showed that the newly developed assay is a reliable non-electrophoretic method in identifying potent DNA ligase inhibitors. The feasibility of the assay was tested in screening a collection of whole cell mass extracts, obtained from a natural product library from Basidiomycetes, in 96-well format. A novel single DNA ligase was identified, expressed and characterised from Trichomonas vaginalis (TV), a pathogenic protozoan parasite. Protein sequence analysis of TV DNA ligase indicates a strong sequence similarity to DNA ligase I homologues. The activity of recombinant TV DNA ligase I (TVlig) was investigated using protein expressed in E.coli cells. The TVlig gene product is 76 kDa and showed optimal ligation activity on a nicked DNA substrate at pH 7-8 in the presence of 1 mM ATP and (8- 20) mM MgCl2 at 30-38oC. The inhibition of the only DNA ligase present in T. vaginalis might suggest for a rational approach to stop replication and hence propagation of the parasite during infection.","abstract_has_math":false,"creators":["Kaur, L."],"institution":"University of Westminster","degree_name":null,"degree_level":"PhD thesis","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2009,"date_issued":"2009","date_published":"2009","updated_at":"2026-07-24T06:02:06Z","subjects":[],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["oai:westminsterresearch.westminster.ac.uk:90xxw"],"render_values":[{"text":"oai:westminsterresearch.westminster.ac.uk:90xxw","href":null,"code":true}]}]},"links":{"outbound_url":"https://doi.org/10.34737/90xxw","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Kaur, L."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2009"]},{"key":"dc:date.issued","label":"Date","values":["2009"]},{"key":"dc:publisher","label":"Institution","values":["University of Westminster"]},{"key":"dc:publisher.department","label":"Dc Publisher Department","values":["School of Life Sciences"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Westminster"]},{"key":"dc:relation","label":"Dc Relation","values":["https://westminsterresearch.westminster.ac.uk/item/90xxw/developing-as-assay-to-screen-inhibitors-for-various-atp-dependent-ligases"]},{"key":"dc:relation.isreferencedby","label":"Dc Relation Isreferencedby","values":["https://westminsterresearch.westminster.ac.uk/item/90xxw/developing-as-assay-to-screen-inhibitors-for-various-atp-dependent-ligases"]},{"key":"dc:type","label":"Dc Type","values":["Thesis or dissertation"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["PhD thesis"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["oai:westminsterresearch.westminster.ac.uk:90xxw"]},{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.34737/90xxw"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://westminsterresearch.westminster.ac.uk/download/1c85bdce8720333bc6e0b0c4f3bf79571d828ae5e20c4f073d9fbde0e8e7d81c/4295951/Loveleen_Kaur_ADDED.pdf"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["DNA ligases (EC.6.5.1.1) are key enzymes that catalyze the formation of phosphodiester bonds at single-stranded or double-stranded breaks between adjacent 5’-PO4 and 3’-OH groups of DNA. These enzymes are essential guardians of genomic integrity and have recently been drawing a lot of attention as novel therapeutic targets in anti-bacterial and anticancer therapies. A novel, non-electrophoretic assay method, based on the strength of interaction of the oligonucleotides with Q-sepharose (a strong anion exchanger), was developed to screen inhibitors of DNA ligases from natural product pools as well as chemical libraries. The binding affinities to Q-sepharose resin of a nicked DNA substrate (created from a 30-mer hairpin oligonucleotide and complementary 32P-labelled 6-mer oligonucleotide) and its sealed, ligated product (36-mer) were determined. Initial optimisation studies were performed with T4 DNA ligase, PBCV-1 DNA ligase and a catalytically active form of human DNA ligase I in the presence of doxorubicin (inhibitor of ATP-dependent ligases). These results when analysed in parallel between the conventional electrophoretic assay and the labelled nick-sealing assay showed that the newly developed assay is a reliable non-electrophoretic method in identifying potent DNA ligase inhibitors. The feasibility of the assay was tested in screening a collection of whole cell mass extracts, obtained from a natural product library from Basidiomycetes, in 96-well format. A novel single DNA ligase was identified, expressed and characterised from Trichomonas vaginalis (TV), a pathogenic protozoan parasite. Protein sequence analysis of TV DNA ligase indicates a strong sequence similarity to DNA ligase I homologues. The activity of recombinant TV DNA ligase I (TVlig) was investigated using protein expressed in E.coli cells. The TVlig gene product is 76 kDa and showed optimal ligation activity on a nicked DNA substrate at pH 7-8 in the presence of 1 mM ATP and (8- 20) mM MgCl2 at 30-38oC. The inhibition of the only DNA ligase present in T. vaginalis might suggest for a rational approach to stop replication and hence propagation of the parasite during infection."]},{"key":"dc:description.abstract","label":"Abstract","values":["DNA ligases (EC.6.5.1.1) are key enzymes that catalyze the formation of phosphodiester bonds at single-stranded or double-stranded breaks between adjacent 5’-PO4 and 3’-OH groups of DNA. These enzymes are essential guardians of genomic integrity and have recently been drawing a lot of attention as novel therapeutic targets in anti-bacterial and anticancer therapies. A novel, non-electrophoretic assay method, based on the strength of interaction of the oligonucleotides with Q-sepharose (a strong anion exchanger), was developed to screen inhibitors of DNA ligases from natural product pools as well as chemical libraries. The binding affinities to Q-sepharose resin of a nicked DNA substrate (created from a 30-mer hairpin oligonucleotide and complementary 32P-labelled 6-mer oligonucleotide) and its sealed, ligated product (36-mer) were determined. Initial optimisation studies were performed with T4 DNA ligase, PBCV-1 DNA ligase and a catalytically active form of human DNA ligase I in the presence of doxorubicin (inhibitor of ATP-dependent ligases). These results when analysed in parallel between the conventional electrophoretic assay and the labelled nick-sealing assay showed that the newly developed assay is a reliable non-electrophoretic method in identifying potent DNA ligase inhibitors. The feasibility of the assay was tested in screening a collection of whole cell mass extracts, obtained from a natural product library from Basidiomycetes, in 96-well format. A novel single DNA ligase was identified, expressed and characterised from Trichomonas vaginalis (TV), a pathogenic protozoan parasite. Protein sequence analysis of TV DNA ligase indicates a strong sequence similarity to DNA ligase I homologues. The activity of recombinant TV DNA ligase I (TVlig) was investigated using protein expressed in E.coli cells. The TVlig gene product is 76 kDa and showed optimal ligation activity on a nicked DNA substrate at pH 7-8 in the presence of 1 mM ATP and (8- 20) mM MgCl2 at 30-38oC. The inhibition of the only DNA ligase present in T. vaginalis might suggest for a rational approach to stop replication and hence propagation of the parasite during infection."]},{"key":"dc:title","label":"Title","values":["Developing as assay to screen inhibitors for various ATP-dependent ligases"]}]}],"canonical_facts":{"dc:creator":["Kaur, L."],"dc:date":["2009"],"dc:date.issued":["2009"],"dc:description":["DNA ligases (EC.6.5.1.1) are key enzymes that catalyze the formation of phosphodiester bonds at single-stranded or double-stranded breaks between adjacent 5’-PO4 and 3’-OH groups of DNA. These enzymes are essential guardians of genomic integrity and have recently been drawing a lot of attention as novel therapeutic targets in anti-bacterial and anticancer therapies. A novel, non-electrophoretic assay method, based on the strength of interaction of the oligonucleotides with Q-sepharose (a strong anion exchanger), was developed to screen inhibitors of DNA ligases from natural product pools as well as chemical libraries. The binding affinities to Q-sepharose resin of a nicked DNA substrate (created from a 30-mer hairpin oligonucleotide and complementary 32P-labelled 6-mer oligonucleotide) and its sealed, ligated product (36-mer) were determined. Initial optimisation studies were performed with T4 DNA ligase, PBCV-1 DNA ligase and a catalytically active form of human DNA ligase I in the presence of doxorubicin (inhibitor of ATP-dependent ligases). These results when analysed in parallel between the conventional electrophoretic assay and the labelled nick-sealing assay showed that the newly developed assay is a reliable non-electrophoretic method in identifying potent DNA ligase inhibitors. The feasibility of the assay was tested in screening a collection of whole cell mass extracts, obtained from a natural product library from Basidiomycetes, in 96-well format. A novel single DNA ligase was identified, expressed and characterised from Trichomonas vaginalis (TV), a pathogenic protozoan parasite. Protein sequence analysis of TV DNA ligase indicates a strong sequence similarity to DNA ligase I homologues. The activity of recombinant TV DNA ligase I (TVlig) was investigated using protein expressed in E.coli cells. The TVlig gene product is 76 kDa and showed optimal ligation activity on a nicked DNA substrate at pH 7-8 in the presence of 1 mM ATP and (8- 20) mM MgCl2 at 30-38oC. The inhibition of the only DNA ligase present in T. vaginalis might suggest for a rational approach to stop replication and hence propagation of the parasite during infection."],"dc:description.abstract":["DNA ligases (EC.6.5.1.1) are key enzymes that catalyze the formation of phosphodiester bonds at single-stranded or double-stranded breaks between adjacent 5’-PO4 and 3’-OH groups of DNA. These enzymes are essential guardians of genomic integrity and have recently been drawing a lot of attention as novel therapeutic targets in anti-bacterial and anticancer therapies. A novel, non-electrophoretic assay method, based on the strength of interaction of the oligonucleotides with Q-sepharose (a strong anion exchanger), was developed to screen inhibitors of DNA ligases from natural product pools as well as chemical libraries. The binding affinities to Q-sepharose resin of a nicked DNA substrate (created from a 30-mer hairpin oligonucleotide and complementary 32P-labelled 6-mer oligonucleotide) and its sealed, ligated product (36-mer) were determined. Initial optimisation studies were performed with T4 DNA ligase, PBCV-1 DNA ligase and a catalytically active form of human DNA ligase I in the presence of doxorubicin (inhibitor of ATP-dependent ligases). These results when analysed in parallel between the conventional electrophoretic assay and the labelled nick-sealing assay showed that the newly developed assay is a reliable non-electrophoretic method in identifying potent DNA ligase inhibitors. The feasibility of the assay was tested in screening a collection of whole cell mass extracts, obtained from a natural product library from Basidiomycetes, in 96-well format. A novel single DNA ligase was identified, expressed and characterised from Trichomonas vaginalis (TV), a pathogenic protozoan parasite. Protein sequence analysis of TV DNA ligase indicates a strong sequence similarity to DNA ligase I homologues. The activity of recombinant TV DNA ligase I (TVlig) was investigated using protein expressed in E.coli cells. The TVlig gene product is 76 kDa and showed optimal ligation activity on a nicked DNA substrate at pH 7-8 in the presence of 1 mM ATP and (8- 20) mM MgCl2 at 30-38oC. The inhibition of the only DNA ligase present in T. vaginalis might suggest for a rational approach to stop replication and hence propagation of the parasite during infection."],"dc:identifier":["oai:westminsterresearch.westminster.ac.uk:90xxw"],"dc:identifier.doi":["https://doi.org/10.34737/90xxw"],"dc:identifier.uri":["https://westminsterresearch.westminster.ac.uk/download/1c85bdce8720333bc6e0b0c4f3bf79571d828ae5e20c4f073d9fbde0e8e7d81c/4295951/Loveleen_Kaur_ADDED.pdf"],"dc:publisher":["University of Westminster"],"dc:publisher.department":["School of Life Sciences"],"dc:publisher.institution":["University of Westminster"],"dc:relation":["https://westminsterresearch.westminster.ac.uk/item/90xxw/developing-as-assay-to-screen-inhibitors-for-various-atp-dependent-ligases"],"dc:relation.isreferencedby":["https://westminsterresearch.westminster.ac.uk/item/90xxw/developing-as-assay-to-screen-inhibitors-for-various-atp-dependent-ligases"],"dc:title":["Developing as assay to screen inhibitors for various ATP-dependent ligases"],"dc:type":["Thesis or dissertation"],"dc:type.qualificationlevel":["PhD thesis"]},"updated_at":"2026-07-24T06:02:06Z"}