9/21/2010 Replication is a self-multiplication to produce a new product which generates itself In molecular level, DNA is the only molecule that is able to replicate (except the RNA molecule of virus) DNA Replication : DNA synthesisDNA = Polynucleotide The Basic Process of Replication: polymerization Assembling the nucleotides into a long chain/strand Nucleotide is the precursor of the DNA synthesis The Precursor : dATP. dGTP, dCTP, dTTP Triphosphate form: synthsized from nucleotide ribose phosphate GDPdGDPdGTP CDP ADP UDP dCDP dADP dUDP dCTP dATP dUMP Kinase phosphatase Ribonucleotide reductase Thymidylate synthetase dTMP Kinase dTTP By Aris Tj 1

2 9/21/2010 The Semi Conservative Model of the DNA Replication new Old strand Old strand new Old strand Conservative Semi conservative diversive The Proof of Semi Conservative Model Matthew Meselson and Franklin Stahl generation Generation-0 15 N Generation-1 14 N- 15 N 14 N 14 N- 15 N Generation-2

9/21/ The Model Of Replication A. Semiconservative In each forming of the DNA double helix molecule, only one strand that is newly generated, meanwhile the other strain is derived from the old strand B. The Origin of Replication (Ori) Only DNA molecule has a replicable Ori. C. The DNA synthesis moves in one or two directions in the growing of 5-3 DNA will be synthesized from the Ori and further moves bi-directionally The new nucleotide is added in 3’OH end D. Replication is moved gradually REPLICATION IS ONLY STARTED IN ORI SITE Only DNA molecule has a replicable Ori Ori site is a DNA sequence that is performed as the signal for DNA polymerase enzyme to start the replication In E.coli, there is one protein (DnaA) which functional to recognize the Ori site in replication DNA from alien species is able to replicate inside the cell of the other species, if only the DNA polymerase from the inhabited cell is capable to recognize the Ori site of the alien species The quantity of Ori site in the DNA is vary – Bacterial chromosome : 1 (one) – Eucaryote chromosome : many – F-Plasmid : Ori-T and Ori-V

9/21/2010 ORI SITE IN REPLICATION PROCESS Ori Bakteri The movement of replication fork Ori The direction of the opening of helix twist 4 The rotation of the twist opening

9/21/2010 Protein dan Enzim yang terlibat 1. The opening of double heliks –––––––– DnaA: initiator protein, building up the primosome (priming complex) DnaB (Helikase ): unveiling the double heliks DnaC: taking up the DnaB to the replication complex DNA Girase: supercoiling, curing the tension in the site of replication branching – Protein SSB: preventing the single strand in the replication fork for joining its complement strand to form the double helix 2. Sintesis Utas Baru – DnaG (Primase) ~ RNA polimerase: synthesizes the RNA primer – DNA polimerase III : synthesizes the polymerization of the new strand DNA – DNA polimerase I : Fills up the gap between two Okazaki fragments and discards the RNA primer – DNA ligase : joining two Okazaki fragments Steps of Replication: 1. Initiation : recognition of Ori (origin of replication) by protein complex 2. Elongation: synthesis of polynucleotide by protein complex called replisome 3. Termination 5

5’TTATCCACA3’ 5’GATCTNTTNTTTT3’ 9/21/2010 Initiation : Proteins involved e.g.: DnaA, DnaB, DnaC, DnaG primase, SSB OriC (Origin of chromosomal replication) dnaA Boxes (DnaA recognition sites DnaB helicase SSB ( Single Strand Binding Protein = helix destabilizing protein) keep the helix from re-forming DnaG primase synthesizes RNA primers, initiating replication in both direction, helped by primosome ( complex of DnaA+DnaB+other proteins ) 5’ 6 With in Ori C: - similar sequences of 9 bases are repeated for times (1, 2, 3, and 4): called DnaA boxes - region 13 base pairs long with a higher than average AT base pairs frequently are repeated three times (L, M, R) The first step of the forming of replication fork is the binding of DnaA in dna boxes DnaA proteins bind to dnaA boxes and opens helix at 13 mers (helix opened partially) DnaC helps the DnaB helicase to bind

9/21/2010 Elongation: synthesis of polynucleotide DNA polymerase III assembles the nucleotides in 3’ –end of RNA primer, for further forms the new DNA strand. The forming of DNA molecule by DNA polymerase III will be stopped if the polymerization reaches up the upcoming RNA primer DNA polymerase I replaces the role of DNA polymerase III: continuing the elongation of DNA strand while discarding the upcoming RNA primer. DnaB helicase opens the helix, therefore the replication fork is moving forward Primer RNA helicase Leading Strand : continously synthesized Lagging strand (Fragmen Okazaki): uncontinously synthesized By Aris Tj 7

LiarMutan Liar 9/21/2010 Errors in nucleotides assembling may happen in DNA replication (errors in nucleotide assembling= “mismatch”) Errors may happen, due to the pairing base is occurred in tautomers form, which different to its normal form. If the replication is completed, the tautomer form will be reformed to its normal form. Mutation will be occurred, if the errors are uncorrected and will be genetically inheritable In order to diminish the errors in replication, cells are equipped with: 1. editing function: correcting the errors during replication 2. Mismatch repair system: correcting the errors after the replication is fully completed Editing Function The editing function may be carried up by DNA polymerase or the other proteins Editing protein (protein for editing purpose) will check and recognize the errors in base pairing and further stop the DNA polymerization process, discard the last added nucleotide The elongation of DNA will be continued if the errors are completely discarded and subsequently replaced by the correct nucleotide. 8

3’ 5’  ’ 5’ Direction of 5’ DNA synthesis: 3’ 3’ Direction of editing by 3’ exonuclease: 5’ 3’ 9/21/2010 The cell reduces mistakes during replication through editing functions Sometime these editing functions are performed by separate proteins, and sometime are part of the DNA polymerase Named “editing proteins” because they go back over the newly replicated DNA looking for mistake, recognizing and removing incorrectly inserted bases Because the DNA chain grows in 5’ to 3’ direction, the last nucleotide added is at the 3’ end. The enzyme activity that remove this nucleotide is therefore called a 3’ exonuclease 3’  5’ : By Aris Tj Bacterial DNA Polymerases scrutinize the base pair at the end of the growing chain and excise the nucleotide added in the case of a misfit In many organisms, include virus, the activity of exonuclease 3’-5’ for editing purpose is part of the DNA polymerase I itself 9

3’ DNA 3” 5” Synthesize of RNA Primer DNA pol DNA polymerase pol Insertion of ribonucleotides (NTP) rather than deoxyribonucleotides (dNTP) during the synthesize of RNA primer causes a distortion. Thus distortion activates the editing protein DnaQ DnaG primase 5” 5’ DNA ’3 helicase 3’ 9/21/2010 Editing protein recognize the mismatch because the mispairing causes a minor distortion in the structure of the double-stranded helix of the DNA In E coli the 3’ exonuclease editing function is encoded by the dnaQ gene. This gene product travel along the DNA with DNA polymerase during replication 3’ By Aris Tj DNA polymerase I has also an activity 5’ exonuclease (the direction of editing is 5’  3’). This activity play a role in : 1. Removing RNA primer (membuang primer RNA yang ada di depannya ketika sedang memanjangkan utas DNA) 5’ Sintesis DNA : 3’ 3’5’ 3’ 5’ 3’ 5’3’ 5’ 3’ 5’3’5’ 3’ 5’ Ligase By Aris Tj 10

T 9/21/ Replacing part of a pre-existing strand of duplex DNA (by Nick translation) with newly synthesized material C