OUP accepted manuscript
Autor: | Horam Soyar, Mukesh Pasupuleti, Kishore K. Srivastava, Taran Khanam, Ravi Sankar Ampapathi, Ravishankar Ramachandran, Kunzes Dolma, Ankita Shukla, Mohammad Afsar, Sanjay Kumar, Faiyaz Alam |
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Rok vydání: | 2020 |
Předmět: |
Exonuclease
chemistry.chemical_classification 0303 health sciences DNA ligase biology DNA repair Base excision repair DNA Ligases Molecular biology DNA-(apurinic or apyrimidinic site) lyase 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine chemistry Genetics 3'-5' Exonuclease biology.protein 030217 neurology & neurosurgery DNA 030304 developmental biology |
Zdroj: | Nucleic Acids Research. |
ISSN: | 1362-4962 0305-1048 |
Popis: | Class-II AP-endonuclease (XthA) and NAD+-dependent DNA ligase (LigA) are involved in initial and terminal stages of bacterial DNA base excision repair (BER), respectively. XthA acts on abasic sites of damaged DNA to create nicks with 3'OH and 5'-deoxyribose phosphate (5'-dRP) moieties. Co-immunoprecipitation using mycobacterial cell-lysate, identified MtbLigA-MtbXthA complex formation. Pull-down experiments using purified wild-type, and domain-deleted MtbLigA mutants show that LigA-XthA interactions are mediated by the BRCT-domain of LigA. Small-Angle-X-ray scattering, 15N/1H-HSQC chemical shift perturbation experiments and mutational analysis identified the BRCT-domain region that interacts with a novel 104DGQPSWSGKP113 motif on XthA for complex-formation. Isothermal-titration calorimetry experiments show that a synthetic peptide with this sequence interacts with MtbLigA and disrupts XthA-LigA interactions. In vitro assays involving DNA substrate and product analogs show that LigA can efficiently reseal 3'OH and 5'dRP DNA termini created by XthA at abasic sites. Assays and SAXS experiments performed in the presence and absence of DNA, show that XthA inhibits LigA by specifically engaging with the latter's BRCT-domain to prevent it from encircling substrate DNA. Overall, the study suggests a coordinating function for XthA whereby it engages initially with LigA to prevent the undesirable consequences of futile cleavage and ligation cycles that might derail bacterial BER. |
Databáze: | OpenAIRE |
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