The large subunit of Leishmania topoisomerase I functions as the 'molecular steer' in type IB topoisomerase.
Kinetoplastid topoisomerase IB is an unusual bisubunit enzyme where reconstitution of the large (LdTOPIL or L) and small (LdTOPIS or S) subunits shows functional activity. It is yet to be deciphered whether one subunit or both navigate the heterodimer to its cellular DNA targets. Tethering a specific DNA-binding protein to topoisomerase I alters its site specificity. The chimeric constructs UMSBP-LdTOPIL/S or U-L/S (fusion of UMSBP to the N-terminus of L and reconstituted with S) and LdTOPIL/UMSBP-LdTOPIS or L/U-S (fusion of UMSBP to the N-terminus of S and reconstituted with L) exhibit relaxation activity. Only U-L/S shows altered site specificity and enhanced DNA-binding affinity for the universal minicircle sequence (UMS) containing substrate. This proves that L alone serves as the 'molecular steer' for this heterodimer. Reconstituted U-L/S also induces cleavage close to UMS and causes minicircle linearization. The differential properties of the reconstituted chimeras U-L/S and L/U-S reveal the structural and functional asymmetry between the heterodimer. Therefore this study helps in a better understanding of the mechanistic details underlying topoisomerization by this bi-subunit enzyme.
BoseDasgupta
Somdeb
S
Department of Molecular Parasitology, Indian Institute of Chemical Biology, Kolkata-700032, India.
Ganguly
Agneyo
A
Das
Benu Brata
BB
Roy
Amit
A
Khalkho
Neeta V M
NV
Majumder
Hemanta K
HK
D000818:Animals; D016880:Anisotropy; D001483:Base Sequence; D002166:Camptothecin / Q000494:pharmacology; D003001:Cloning, Molecular; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004278:DNA, Superhelical / Q000737:chemistry / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004795:Enzyme Stability / Q000187:drug effects; D007893:Leishmania donovani / Q000201:enzymology*; D011189:Potassium Chloride / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D021122:Protein Subunits; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D011189:Potassium Chloride; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin