At this moment putting down a few explicit words of thanks in my acknowledgement is a very difficult task for me since the depth of my feelings sometimes is too tough to be arrested in few words. First and foremost I take this opportunity to thank my supervisor Professor J.P.Khurana whose magnanimous attitude and generous approach that gave me judicious guidelines with humane touch to accomplish my project.
I will not lag behind in paying regards to my Honorable ‘s Head of the centre for Biotechnology, Professor P.S Srivastava whose headship combined with scholarship and guardianship not only impacted knowledge but awakened interest for the pursuit excellence. I also wish to express my grateful to my esteemed faculty Prof.S.K. Jain, Dr M.Z. Abdin, Dr.Fahrat Afrin, Dr. Depsika Pande, Dr. Farha Anjum and Dr. Sandeep Das for their valuable suggestions, support and encouragement.
I am highly indebted to Mr.Mukesh Jain for inculcating me all those qualities, which make him so meticulous, and for training my work and me untiring at each and every stage got an appreciable order due to helping hands provided by Mr.Mukesh jain. And he deserves special thanks for helping a great deal in my concluding Experiment. Next I express my profound sense gratitude to Dr. Ritu, Mithu, Jithendra Thakur, and Laju Paul for their noble guidance and effective co-operative.
The two polynucleotide of the parent helix are wound round one another, this means that progression of the replication require the double helix not to just to be unzipped but also to be un wound. This infects a significant problem if we consider that the E.coli DNA molecule is 4000k.b or 400000 turns of helix in length and must be replicated in twenty minute. The implication that the double helix is rotating at a rate of 6500rpm! This is so inconceivable that for many years molecular biologist s sought solution that avoid unwinding around 1979 things became so desperate that double helix was incorrect and in fact the polynucleotide in double stranded DNA molecule are laid side by side and not wound round each other. Fortunately a group of enzyme that solved the topological problem was eventually discovered.
DNA topoisomerases are a class of enzyme involved in the regulation of DNA super coiling. DNA topoisomerases fall in to two classes Type I and Type II. Both unwind DNA molecules without actually rotating the double helix. Type I topoisomerases change the degree of super coiling of DNA by causing single stranded break and religation, where type II topoisomerases cause double stranded break. Different roles of topoisomerase I and II indicate an opposing pair of roles in the regulation of DNA super coiling. Both activities are especially crucial during DNA transcription and replication when the DNA helix must be unwound to allow proper function of large enzymatic machinery and topoisomerases have indeed been shown to maintain both transcription and replication.
DNA TOPOISOMERASE I
Structure; The determination of crystal structure of a 67kDa and terminal fragment of E.coli topoisomerase I represents how these enzymes function. Str-1 The 590 aa N-terminal fragment observed in the structure corresponds to the cleavage/strand passage domain. E.coli topo I contains four domains of protein. This mechanism has been referred to as enzyme-bridging model for DNA relaxation.
The crystal structure of the 67kDa fragment of E.coli enzyme suggests how such reaction occurs. The active site tyr319 is buried in the structure. It is proposed that as the single stranded DNA binds to the cleft, domain III undergoes conformational adjustment to place the nucleophilic O-4 oxygen of the tyrosine side chain in a position to attack the phosphate. After the cleavage the active site tyr319 is covalently bound to the 5′ phosphate on one end of the cleaved and the other end is proposed to occupy a nucleotide binding site at the end of cleft in
Immediately upon cleavage domain III that is holding onto the 5’ end of the broken strand, lifts away from domain I to create a gap through which is passed either intact strands. Once the intact strands have moved into the hole of the torus, the clamp closes and the cleaved strand is religated. The protein must then opened and closed second time to release the passed strand to complete the cycle. Once reset, the enzyme can dissociate from the DNA or act processively to carry out another cycle of strand passage.