L3: DNA Replication PDF

L3: DNA Replication There is no short cut to success and substitute for hard work.. L3: DNA Replication L3: DNA Replication 1  L3: DNA Replication 2 How β-clamp/PCNA sliding clamp makes DNA polymerase processive?  L3: DNA Replication...

L3: DNA Replication There is no short cut to success and substitute for hard work.. L3: DNA Replication L3: DNA Replication 1  L3: DNA Replication 2 How β-clamp/PCNA sliding clamp makes DNA polymerase processive?  L3: DNA Replication 3 What is clamp loader? Prokaryotes: The γ, δ, and δ’ subunit of DNA polymerase III Eukaryotes: The protein Replication Factor C RFC 1-5: Five-subunit hetero-pentamer Clamp loading is an ATP-dependent process Clamp loader and DNA polymerase compete for the same C-terminal face of the clamp  L3: DNA Replication 4 DNA polymerase III holoenzyme: Includes four enzymes- 3 copies of the DNA Pol III core enzyme and one copy of the sliding clamp holder L3: DNA Replication 5 L3: DNA Replication 6 L3: DNA Replication 7 L3: DNA Replication 8 L3: DNA Replication 9 L3: DNA Replication 10 L3: DNA Replication 11  L3: DNA Replication 12 DNA ligase joins together Okazaki fragments on the lagging strand during DNA synthesis  L3: DNA Replication 13 How Okazaki fragment are matured to a continuous DNA strand in Eukaryotes? Rnase H2 , Dna2 and Fen1 RNaseH2 is a family of non-sequence-specific endoribonuclease enzymes that catalyze the cleavage the phosphodiester bonds of RNA which is hybridized to DNA DNA replication helicase/endonuclease 2 (Dna2) Fen 1 is structure-specific nuclease with 5'-flap endonuclease and 5'-3' exonuclease  L3: DNA Replication 14 Different pathways for removal of primer in eukaryotes  L3: DNA Replication 15 Why topoisomerase are essential for DNA replication?  L3: DNA Replication 16 Type IA and IB topoisomerases make single stranded cut on either of the two strands. Topoisomerase II cleave both strands, in a reaction that requires ATP as energy.  L3: DNA Replication 17 Bacterial DNA gyrase introduces negative supercoiling Campothecin, norflaxin, flaxin and ciprofloxacin are widely used antibiotic drugs as they inhibit bacterial topoisomerase II (Gyrase).  L3: DNA Replication 18 Eukaryotic Topoisomerase Inhibitors Doxorubicin, irinotecan, etoposide are often used in treatment of colorectal, lung and ovarian cancers.  L3: DNA Replication 19 DNA Replication in Bacteria What is the replicon model? Francois Jacob, Sydney Brenner, and Jacques Cuzin The entire DNA that is replicated from an origin of replication is collectively called as replicon Initiator proteins: AAA+ ATP-binding motif  L3: DNA Replication 20 What is nature of replicator sequences? Includes a binding site for the initiator Stretch of AT-rich DNA E. coli has a single replicator sequence – Ori C Archaebacteria Sulfolobus have 3 origins  L3: DNA Replication 21 What is function of the Initiator Proteins? 1. Origin Selection 2. Activation 3. Unwinding E. coli initiator protein DnaA L3: DNA Replication 22 L3: DNA Replication 23 L3: DNA Replication 24  L3: DNA Replication 25 Primosome Complex Association of helicase, primase and SSB. Primase is not tightly associated with the fork. At an interval 1/sec, primase associates with the helicase and SSB-coated ssDNA and synthesizes a new RNA primer on lagging strand. The interaction between the DNA helicase and primase is weak But strongly stimulates primase function(about 1000-fold). A weaker interaction result in longer Okazaki fragments.  L3: DNA Replication 26 Interactions between Replication Fork Proteins Form the E. coli Replisome Tau subunit of the clamp loader holds the helicase and the DNA Pol III holoenzyme. The activity of the helicase movement is increased 10-fold. This prevents the helicase from “running away” from the DNA Pol III holoenzyme L3: DNA Replication 27 L3: DNA Replication 28  L3: DNA Replication 29 Type II Topoisomerases (gyrase) Are Required to Separate Daughter DNA Molecules L3: DNA Replication 30 In E. coli grown under nutrient rich conditions, replication of entire genome takes about 40 min., yet it can divide every 20 min L3: DNA Replication L3: DNA Replication L3: DNA Replication