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REPLICATION M.Prasad Naidu MSc Medical Biochemistry, Ph.D,. \
Introduction • Besides maintaining the integrity of DNA sequences by DNA repair, all organisms must duplicate their DNA accurately before every cell division. • DNA replication occurs at polymerization rates of about 500 nucleotides per second in bacteria and about 50 nucleotides per second in mammals. • Clearly, the proteins that catalyze this process must be both accurate and fast. • Speed and accuracy are achieved by means of a multienzyme complex that guides the process and constitutes an elaborate "replication machine."
Replication occurs in5’ to 3’ direction only. • Replication is simultaneous on both strands. • Replication is bidirectional. • Replication obeys base pair rule • Replication results in 2 daughterDNA strands. • Each daughter DNA strand has one • parent strand and one complementary • strand synthesized newly. Hence this • Replication is semi-conservative. • Held by phospho-di-ester bonds • and Hydrogen bonds
CELL - CYCLE • Cell cycle is a sequence of events that occur in a cell during cell division. • It results in formation of 2 identical daughter cells. • Duration of cell cycle varies from cell to cell. • It occurs in 4 phases G1 PHASE [ gap-1] S PHASE [synthetic] G2 PHASE [gap-2] M PHASE [ mitotic]
Cell- cycle • G1 phase ; Preparative phase for DNAsynthesis. All cellular components replicate except DNA . Cell size increases. Any damage to DNA is detected. • S phase ;DNA replication takes place. • G2 phase; Prepares for cell division and spindle formation. Any damage to DNA is detected. • M phase;Cell undergoes cell division . It includes prophase ,metaphase, anaphase ,and telophase. After mitosis cell may continue cycle by re-entering into G1 or enter G0 and remain dormant or leads to cell death
Models for DNA REPLICATION • These are many hypothesis to explain the process of replication. They are • Conservative model • Semi conservative model • Dispersive model
DNA-Replication Requirements 1.Deoxyribonucleotides [ dATP, dGTP, dCTP, dTTP ] 2.Template DNA strand [parent strand] 3.RNA primer 4.EnzymesDNA polymerase Primase Helicase DNA Ligase Topo-isomerases Single Strand Binding Proteins.
Single strand binding protein (SSBP ) Binds to ssDNA Has two function 1. prevents reannealing , thus providing ss template required by polymerases 2. protects ssDNA from nuclease activity Show cooperative binding
Helicases • Separate the ds DNA to ss DNA by dissolving the hydrogen bonds holding the two strands together • These separates dsDNA at physiological temperature • ATP dependent • At least 9 helicases have been described in E coli • Of which DNA binding protein A, B , C ( Dna A, Dna B, Dna C ) are most important • Initial separation is by Dna A • Continued further by Dna B ( major strand separating protein acts bidirectionally ) • Dna C is required for loading Dna B at site of replication
Primase: • Primase is a specilised RNA polymerase • It synthesis a short strech of RNA in 5’ 3’ direction on a template running in 3’ 5’ direction. • An RNA primer, about 100-200 nucleotides long, is synthesized by the RNA primase. • The RNA primer is removed by DANP, using exonuclease activity and is replaced with deoxyribo nucleotides by DNAP
DNA Ligases DNA ligases close nicks in the phosphodiester backbone of DNA. Two of the most important biologically roles of DNA ligases are: 1. Joining of Okazaki fragments during replication. 2. Completing short-patch DNA synthesis occurring in DNA repair process. • There are two classes of DNA ligases: • The first uses NAD+ as a cofactor and only found in bacteria. • The second uses ATP as a cofactor and found in eukaryotes, viruses and bacteriophages.
DNA Ligase Mechanism • The reaction occurs in three stages in all DNA ligases: • Formation of a covalent enzyme-AMP intermediate linked to a lysine side-chain in the enzyme. • Transfer of the AMP nucleotide to the 5’-phosphate of the nicked DNA strand. • Attack on the AMP-DNA bond by the 3’-OH of the nicked DNA sealing the phosphate backbone and resealing AMP.
SUPERCOILS • As two strands unwind ,they result in the formation of positive supercoils ( super twists ) in the region of DNA ahead of replication fork. • Accumulation of these supercoils interfere with further unwinding of ds DNA. • This problem is solved by the enzyme Topoisomerases. • These catalyze the interconvertion of topoisomers of DNA
Catalyze in a three step process 1. cleavage of one or both strands of DNA 2. passage of a segment of DNA through this break 3. resealing of the DNA Two types of topoisomerases are present DNA which different in the linking numer Linking number = (Twist +Wreth) 3 dimentional -type I topoisomerases -type II topoisomerases
Topoisomerases I • Reversibly cut one strand of double helix • Have both nuclease ( strand cutting ) & ligase ( strand resealing ) • Donot require ATP ,rather use the energy released by phosphodiester bond cleavage to reseal the nick • Removes only negative super coils • Ex : bacteria
Topoisomerases II ( DNA gyrase ) • Heterodimer with 2 swivelase & 2 ATPase subunits • Swivelase subunit catalyzes trans esterification reaction that breaks & reforms the phosphodiester backbone • ATPase subunit hydrolyzes ATP to trigger conformational changes that allow a double helix to pass through the transient gap • Possitive super coiled
DNA polymerases • These are the enzymes responsible for the polymerisation of deoxyribo nucleosides, triphosphates on a DNA template strand to form a new complementary DNA strand. • In prokaryotes based on site and conditions of action. They are divided into 3 types: I II III.
Common properties: • All polymerases can synthesis a new strand of DNA in 5’ to 3” direction. On a template strand which is running in 3’to 5’ direction. • They also show Exo nuclease activity ( it cleaves the end terminals of DNA) in 3’to 5’ direction. • All DNA polymerases cannot initiate the process of replication on their own. This is the basic defect of DNAP synthesis of new strand .
Replication There are three phases of replication 1. Initiation 2. Elongation 3. Termination
Steps in DNA-replication 1.Recognition of origin of replication and Un- winding of double stranded DNA 2.Formation of replication bubbles with 2 replication forks for each replication bubble. 3.Initiation and elongation of DNA strand. 4.Termination and Reconstitution of chromatin structure.
InitiationofDNA-REPLICATION 1.Identification of the origins of replication. • The origin of replication [oriC locus] rich in AT pairs is identified. • A specific protein [Dna A] binds to the oriC and results in unwinding of ds DNA. • Un winding of DNA results in formation of replication bubble with 2 replication forks. • Ss binding proteins binds to DNA to each strand to prevent re-annealing of DNA. • Helicases continues the process of un winding. • Topoisomerases relieve the super coils formed during unwinding.
DNA-replication 2.Fomation of replication fork • replication fork has 4 components 1.helicase[unwinds ds DNA] 2.primase [synthesizes RNA primer] 3.DNApolymerase[synthesizes DNA] 4.ss binding proteins [stabilizes the strand]
2.Elongation of DNA • Requires RNA primer, DNA template , DNAP enzyme and deoxyribonucleotides [dATP,dGTP ,dCTP, dTTP] • DNA polymerase catalyze the stepwiseaddition of deoxyribonucleotides to 31 end of template strand and thus copies the information from the template DNA. • DNAP requires RNA primer to start elongation. • DNAP copies the information from DNA template
2.Elongation of DNA 1.continous synthesis occurs towards the replication fork [leading strand] by DNA polymerase. 2.discontinuous synthesis occurs away from the replication fork in pieces called as okazaki fragments which are ligated by DNA ligase [lagging strand]. It requires multiple RNAprimers.
Okazaki fragments • First demonstrated by Reiji Okazaki • Short fragments of DNA present on the lagging strand resulted by retrograde synthesis. • Okazaki fragments in human cells average about 130 - 200 nucleotide in length • In E coli they are about ten times this.
