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Structure/function analyses indicate novel roles for mycobacterial DnaQ homologs in genome maintenance
Mycobacterial DNA metabolism is of increasing interest as both an underexplored source of new targets for anti-tuberculosis (TB) drug development and for its potential role in the emergence of drug-resistant Mycobacterium tuberculosis strains. However, the redundancy implied by the sizable complemen...
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| Format: | Thesis |
| Language: | English English |
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Department of Pathology
2025
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| _version_ | 1867613225199075328 |
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| access_status_str | Open Access |
| author | Griffault, Dimitri |
| author2 | Warner, Digby |
| author_browse | Griffault, Dimitri Warner, Digby |
| author_facet | Warner, Digby Griffault, Dimitri |
| author_sort | Griffault, Dimitri |
| collection | Thesis |
| description | Mycobacterial DNA metabolism is of increasing interest as both an underexplored source of new targets for anti-tuberculosis (TB) drug development and for its potential role in the emergence of drug-resistant Mycobacterium tuberculosis strains. However, the redundancy implied by the sizable complement of DNA replication and repair pathways complicates investigations of gene function. There are, moreover, multiple examples in mycobacteria of apparent fusion – or hybrid – proteins in which N-and C-terminal domains appear to provide discrete functions. Both challenges apply to the mycobacterial DnaQ homologs – comprising separate DnaQ and DnaQ-UvrC hybrid proteins – which, by analogy to model organisms such as E. coli, have traditionally been assumed to fulfil proofreading roles in DNA replication owing to the presence of conserved exonuclease domains. Phylogenetic analysis of DnaQ-like proteins revealed a unique domain composition specific to the Mycobacterium genus comprising a conserved BRCA1 C Terminus (BRCT) domain in DnaQ. Owing to the presence of the BRCT domain and based on the phenotypes observed in domain-targeted mutants, it appeared that the activity of DnaQ protein (M. tuberculosis Rv3711c; M. smegmatis MSMEG_6275) might be linked to the mycobacterial gyrases that are responsible for DNA negative supercoiling following replication. The phylogenetic analysis also revealed highly conserved nucleotide excision repair (NER) proteins among bacteria; however, some species like Actinobacteria, possess both a canonical UvrC along with a DnaQ-UvrC protein. The mycobacterial DnaQ-UvrC (M. tuberculosis Rv2191; M. smegmatis MSMEG_4259) N- terminal was shown to be structurally very similar to that of DnaQ and its C-terminal to that of UvrC, giving the ability to bind either the β-clamp or the components of the UvrABC system. Opposing phenotypes between DnaQ-UvrC (M. tuberculosis Rv2191; M. smegmatis MSMEG_4259) and uvrC deletion imply a DNA-damage specific NER in mycobacteria. This was further confirmed using a combination of gene knockout, site-directed mutants and CRISPRi of NER genes, namely uvrB and uvrC, in DNA damaging conditions. However, further work is required to elucidate the precise functions of DnaQ and DnaQ-UvrC, and their contribution to genome dynamics in a family of organisms that includes major human and animal pathogens. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/42341 |
| institution | University of Cape Town (South Africa) |
| language | English eng |
| last_indexed | 2026-06-10T12:32:45.765Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2025 |
| publishDateRange | 2025 |
| publishDateSort | 2025 |
| publisher | Department of Pathology |
| publisherStr | Department of Pathology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/42341 Structure/function analyses indicate novel roles for mycobacterial DnaQ homologs in genome maintenance Griffault, Dimitri Warner, Digby DNA Mycobacterial Mycobacterial DNA metabolism is of increasing interest as both an underexplored source of new targets for anti-tuberculosis (TB) drug development and for its potential role in the emergence of drug-resistant Mycobacterium tuberculosis strains. However, the redundancy implied by the sizable complement of DNA replication and repair pathways complicates investigations of gene function. There are, moreover, multiple examples in mycobacteria of apparent fusion – or hybrid – proteins in which N-and C-terminal domains appear to provide discrete functions. Both challenges apply to the mycobacterial DnaQ homologs – comprising separate DnaQ and DnaQ-UvrC hybrid proteins – which, by analogy to model organisms such as E. coli, have traditionally been assumed to fulfil proofreading roles in DNA replication owing to the presence of conserved exonuclease domains. Phylogenetic analysis of DnaQ-like proteins revealed a unique domain composition specific to the Mycobacterium genus comprising a conserved BRCA1 C Terminus (BRCT) domain in DnaQ. Owing to the presence of the BRCT domain and based on the phenotypes observed in domain-targeted mutants, it appeared that the activity of DnaQ protein (M. tuberculosis Rv3711c; M. smegmatis MSMEG_6275) might be linked to the mycobacterial gyrases that are responsible for DNA negative supercoiling following replication. The phylogenetic analysis also revealed highly conserved nucleotide excision repair (NER) proteins among bacteria; however, some species like Actinobacteria, possess both a canonical UvrC along with a DnaQ-UvrC protein. The mycobacterial DnaQ-UvrC (M. tuberculosis Rv2191; M. smegmatis MSMEG_4259) N- terminal was shown to be structurally very similar to that of DnaQ and its C-terminal to that of UvrC, giving the ability to bind either the β-clamp or the components of the UvrABC system. Opposing phenotypes between DnaQ-UvrC (M. tuberculosis Rv2191; M. smegmatis MSMEG_4259) and uvrC deletion imply a DNA-damage specific NER in mycobacteria. This was further confirmed using a combination of gene knockout, site-directed mutants and CRISPRi of NER genes, namely uvrB and uvrC, in DNA damaging conditions. However, further work is required to elucidate the precise functions of DnaQ and DnaQ-UvrC, and their contribution to genome dynamics in a family of organisms that includes major human and animal pathogens. 2025-11-26T08:03:33Z 2025-11-26T08:03:33Z 2025 2025-11-26T08:01:51Z Thesis / Dissertation Doctoral PhD http://hdl.handle.net/11427/42341 en eng application/pdf Department of Pathology Faculty of Health Sciences University of Cape Town |
| spellingShingle | DNA Mycobacterial Griffault, Dimitri Structure/function analyses indicate novel roles for mycobacterial DnaQ homologs in genome maintenance |
| thesis_degree_str | Doctoral |
| title | Structure/function analyses indicate novel roles for mycobacterial DnaQ homologs in genome maintenance |
| title_full | Structure/function analyses indicate novel roles for mycobacterial DnaQ homologs in genome maintenance |
| title_fullStr | Structure/function analyses indicate novel roles for mycobacterial DnaQ homologs in genome maintenance |
| title_full_unstemmed | Structure/function analyses indicate novel roles for mycobacterial DnaQ homologs in genome maintenance |
| title_short | Structure/function analyses indicate novel roles for mycobacterial DnaQ homologs in genome maintenance |
| title_sort | structure function analyses indicate novel roles for mycobacterial dnaq homologs in genome maintenance |
| topic | DNA Mycobacterial |
| url | http://hdl.handle.net/11427/42341 |
| work_keys_str_mv | AT griffaultdimitri structurefunctionanalysesindicatenovelrolesformycobacterialdnaqhomologsingenomemaintenance |