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Structure/function studies of the NAD+-dependent DNA ligase from the poly-extremophile Deinococcus radiodurans reveal importance of the BRCT domain for DNA binding

2023-12Artigo científico Acesso aberto
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dc.contributor.authorFernandes, Andreia
dc.contributor.authorWilliamson, Adele
dc.contributor.authorMatias, Pedro M.
dc.contributor.authorMoe, Elin
dc.contributor.institutionInstituto de Tecnologia Química e Biológica António Xavier (ITQB)
dc.contributor.pblSpringer
dc.date.accessioned2024-04-02T23:51:44Z
dc.date.available2024-04-02T23:51:44Z
dc.date.issued2023-12
dc.descriptionFunding Information: The authors would like to thank the team of Diamond-CCP4 Data Collection and Structure Solution Workshop 2021 for assistance in data processing, especially Dr. Claudia Millán (University of Cambridge) for helping in the MR steps. Beam time and assistance by beamline staff at BL13-XALOC beamline at the ALBA Synchrotron, and beamline ID23 at the European Synchrotron Radiation Facility are gratefully acknowledged. This work was financially supported by: Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020, by national funds (PTDC/BBB-BEP/0561/2014) and a PhD fellowship PD/BD/13548/2018 to AF through FCT—Fundação para a Ciência e a Tecnologia. The BIOTEK2021 programme of the Research Council of Norway (NRC), under Grant No. 247732 supported AW. Funding Information: Open access funding provided by FCT|FCCN (b-on). This work was supported by FCT—Fundação para a Ciência e a Tecnologia, I.P., through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020), research projects PTDC/BBB-BEP/0561/2014, post doc fellowship SFRH/BPD/97493/2013 (EM), and PhD fellowship PD/BD/13548/2018 (AF). The Royal Society of New Zealand (Marsden Fund of New Zealand [18-UOW-034] and Rutherford Discovery Fellowship [RDF-UOW2002] provided support (AW). Publisher Copyright: © 2023, The Author(s).
dc.description.abstractBacterial NAD+-dependent DNA ligases (LigAs) are enzymes involved in replication, recombination, and DNA-repair processes by catalyzing the formation of phosphodiester bonds in the backbone of DNA. These multidomain proteins exhibit four modular domains, that are highly conserved across species, with the BRCT (breast cancer type 1 C-terminus) domain on the C-terminus of the enzyme. In this study, we expressed and purified both recombinant full-length and a C-terminally truncated LigA from Deinococcus radiodurans (DrLigA and DrLigA∆BRCT) and characterized them using biochemical and X-ray crystallography techniques. Using seeds of DrLigA spherulites, we obtained ≤ 100 µm plate crystals of DrLigA∆BRCT. The crystal structure of the truncated protein was obtained at 3.4 Å resolution, revealing DrLigA∆BRCT in a non-adenylated state. Using molecular beacon-based activity assays, we demonstrated that DNA ligation via nick sealing remains unaffected in the truncated DrLigA∆BRCT. However, DNA-binding assays revealed a reduction in the affinity of DrLigA∆BRCT for dsDNA. Thus, we conclude that the flexible BRCT domain, while not critical for DNA nick-joining, plays a role in the DNA binding process, which may be a conserved function of the BRCT domain in LigA-type DNA ligases.en
dc.description.versionpublishersversion
dc.description.versionpublished
dc.format.extent2017931
dc.identifier.doi10.1007/s00792-023-01309-z
dc.identifier.issn1431-0651
dc.identifier.otherPURE: 83700127
dc.identifier.otherPURE UUID: 8a2da2eb-af55-4933-824a-e0598b265d11
dc.identifier.otherScopus: 85171391097
dc.identifier.otherPubMed: 37712998
dc.identifier.otherORCID: /0000-0001-6170-451X/work/156734644
dc.identifier.urihttp://hdl.handle.net/10362/165737
dc.identifier.urlhttps://www.scopus.com/pages/publications/85171391097
dc.language.isoeng
dc.peerreviewedyes
dc.subjectBRCT
dc.subjectDNA ligase A
dc.subjectDNA nick-joining
dc.subjectProtein–DNA binding
dc.subjectX-ray crystallography
dc.subjectMicrobiology
dc.subjectMolecular Medicine
dc.subjectSDG 3 - Good Health and Well-being
dc.titleStructure/function studies of the NAD+-dependent DNA ligase from the poly-extremophile Deinococcus radiodurans reveal importance of the BRCT domain for DNA bindingen
dc.typejournal article
degois.publication.issue3
degois.publication.titleExtremophiles
degois.publication.volume27
dspace.entity.typePublication
rcaap.rightsopenAccess

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