REPLICATION OF DNA 1 Restriction enzyme • Endonuclease – cleave the nt in the middle of DNA molecule • Exonuclease - cleave the nt from the end of DNA molecule 2 The flow of genetic information • Duplication of DNA to produce a new DNA molecule with the same base sequence as original • A necessary process whenever a cell divides to produce daughter cells • Flow of genetic information DNA –RNA-Protein 3 Replication process • A complex process • Ensures fidelity • 3 important challenge 1. Separating the two strands – the double helix must be unwound if they are to be separated. At the same time, the unwound portions must be protected from the action of nucleases that prefer to attack ss DNA. 2. Synthesizing DNA from 5’ end to the 3’end. 3. Guarding against error in replication – ensuring the correct base is added 4 Types of replication • Conservative The parental strands never completely separate After on round replication, one daughter duplex contains only parental strands and the other only daughter strands • Semi conservative The process of unwinding, of the double helical daughter molecules – that is composed of a parental strand and a newly synthesized strand formed from the complementary strand • Theta replication – replication in a circular form -prokaryote 5 Semiconservative replication 6 Steps involved in DNA replication a) Identification of the origins of replication b) Unwinding (denaturation) of dsDNA to provide ssDNA template c) Formation of the replication fork d) Initiation of DNA synthesis and elongation e) Formation of replication bubbles with ligation of the newly synthesized DNA segments f) Proof reading process DNA replication in Prokaryote and eukaryote generally involve the same steps, except in eukaryote the process is more complex due to the presence of histone complex 7 DNA REPLICATION IN E.COLI-FAMOUS MODEL 8 a) Origin of replication • Origin of replication (oriC )- spesific sites where replication starts • Sequence specific DNA binding proteins (O Protein) will bind to ori • Adjacent to ori is A+T region • Binding of O protein lead to local denaturation and unwinding of A+T region 9 b) Unwinding of DNA to form SS DNA which act as template • Interaction of O protein provide a short region of ssDNA essential for initiation of replication • This region – a template for initiation • DNA helicases helps in unwinding of DNA • DNA helices produces nicks in one strand of double helix • Single stranded binding proteins (SSB Proteins) – bind to SS each strand and stabilize the complex and prevents re- annealing 10