Critical determinants for substrate recognition and catalysis in the M. tuberculosis class II AP-endonuclease/3'-5' exonuclease III.
The Mycobacterium tuberculosis AP-endonuclease/3'-5' exodeoxyribonuclease (MtbXthA) is an important player in DNA base excision repair (BER). We demonstrate that the enzyme has robust apurinic/apyrimidinic (AP) endonuclease activity, 3'-5' exonuclease, phosphatase, and phosphodiesterase activities. The enzyme functions as an AP-endonuclease at high ionic environments, while the 3'-5'-exonuclease activity is predominant at low ionic environments. Our molecular modelling and mutational experiments show that E57 and D251 are critical for catalysis. Although nicked DNA and gapped DNA are fair substrates of MtbXthA, the gap-size did not affect the excision activity and furthermore, a substrate with a recessed 3'-end is preferred. To understand the determinants of abasic-site recognition, we examined the possible roles of (i) the base opposite the abasic site, (ii) the abasic ribose ring itself, (iii) local distortions in the AP-site, and (iv) conserved residues located near the active site. Our experiments demonstrate that the first three determinants do not play a role in MtbXthA, and in fact the enzyme exhibits robust endonucleolytic activity against single-stranded AP DNA also. Regarding the fourth determinant, it is known that the catalytic-site of AP endonucleases is surrounded by conserved aromatic residues and intriguingly, the exact residues that are directly involved in abasic site recognition vary with the individual proteins. We therefore, used a combination of mutational analysis, kinetic assays, and structure-based modelling, to identify that Y237, supported by Y137, mediates the formation of the MtbXthA-AP-DNA complex and AP-site incision.
Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Lucknow, Uttar Pradesh 226031, India.
Taran
T
Khanam
Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Lucknow, Uttar Pradesh 226031, India.
Ankita
A
Shukla
Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Lucknow, Uttar Pradesh 226031, India.
Niyati
N
Rai
Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Lucknow, Uttar Pradesh 226031, India. Electronic address: r_ravishankar@cdri.res.in.
Ravishankar
R
Ramachandran
D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase
AP site recognition; Base excision repair; DNA damage; DNA repair; XthA