چرخه سلولي Go

Objectives: To know and explain about:  What are needed for Replication  Template properties  Start site of replication( the origin) Enzymes of Replication  Enzymes of Replication  Protein Factors of Replication  Different Stages of Replication   Simultaneous DNA synthesis in two forks (bidirectional)  Simultaneous DNA synthesis in two strands, (leading & lagging)

Objectives cont.   Replication termination in E. Coli   The fidelity of DNA replication   How to ensure initiation of replication occures only once per cycle ?   How to control the multiple replicons to be activated only once time in a single cell cycle   Comparison of replication in eu and pro   Termination in eu   Toxins and antibiotics that inhibit replication

DNA

DNA

DNA overall view 1’ 2’3’ 4’ 5’ (see, no oxygen at 2’…) Deoxyribose

Replication( overall view) During cell division Whole DNA must replicate During cell division Whole DNA must replicate The DNA double helix unwinds The DNA double helix unwinds The exposed bases bind to free floating nucleotides in the nucleoplasm The exposed bases bind to free floating nucleotides in the nucleoplasm DNA polymerase binds the complimentary nucleotides DNA polymerase binds the complimentary nucleotides

The Permission

Theory of Replication Bacteria grown in 14 N media Bacteria grown in 15 media Transfer to 15 N media for one replication Which one is correct? Let ’ s check !

1- Template ( Double Stranded DNA) 2- Building blocks ( dNTPs & NTPs) 3- Enzymes 4- Energy ( Energy of dNTPs & ATP) 5- Protein Factors What are needed for Replication

The template It is a double stranded DNA It should have a specific sequence (origin) for replication to be started It is replicated in semiconservative manner It is replicated bidirectionaly

Replication: Is it bidirectional or unidirectional / How to prove it By using H3T with two different concentrations it was proved that in most cases it is Bidirectional u Unidirectional Bidirectional

Replication / The origin Replication starts from Origin Replication starts from Origin Origin has specific AT rich sequences Origin has specific AT rich sequences Because of the shape of the Because of the shape of the origin, replicating bacteria DNA is called teta form 4 of 9 mer 3 of 13 mer OriTeta form A minimal Origin sequence is consisted of : 245bp

Enzymes of Replication 1- DNAPs ( DNA synthesis, gap filling) 2- RNAP =primase ( priming DNA synthesis) 3- Helicases ( opening the helix turns) 4- Topoisomerase (removing the superheix turns) 5- Ligase ( sealing the nicks between Okazaki fragments)

Function: 1- 5’ to 3’DNA synthesis 2- 3’ to 5’ Exonuclese activity (proof reading) 3- 5’ to 3’ exonuclese activity just DNAP1 Structure: Is oligomeric and is composed of different subunits Types: DNAP I: fills gaps left by the removal of RNA primer and involves in DNA repair Its Klenow fragment (C-large fragment=68kDa) has 5’-3’ polymerase and 3‘-5’ exonuclease activity DNAP II: Involves in DNA repair DNAP III: Chromosome replicating enzyme 2- Enzymes/a: DNA polyerases

DNA polymerase III holoenzyme is very complex,with many polypepetides 1- major subunits,core 2-minor subunits dsDNA β subunite ε 3 5 exo… n/s α Polymerase activity ז Dimerization of core enzyme

DNA polymerase

I III II 5‘ 3‘ 5‘ 3‘ polymerase Exonuclease ( Proof reading) exonuclease TNO( nt/min) Mass (kDa) Numbers/ cell ??? Bioactivity Gene pol C ＊ pol A pol B DNA Polymerases in Bacteria

DNAPs can not initiate DNA synthesis (no de novo synthesis) and there must be small pre-existing primersDNAPs can not initiate DNA synthesis (no de novo synthesis) and there must be small pre-existing primers The enzyme responsible for primer synthesis is called Primase It synthesize the primer from 5 ’ to 3 ’It synthesize the primer from 5 ’ to 3 ’ It has not exonuclease activityIt has not exonuclease activity It is part of primosomeIt is part of primosome 2- Enzymes/b: Primase( Dna G)

2- Enzymes/c: Topoisomerases Enzymes which relieve stress on the DNA by allowing free rotation around a single or double stranded DNA There are two classes: 1 & 2 Class 2 in bacteria is called gyrase. Gyrase reduces two turn of DNA each time ( convert +ve superhelix into – ve)

2- Enzymes/d: Helicases There are different types of helicases : Dna B Other Helicases (rep protein) Enzyme which catalyze the unwinding and separation ( bearking H-bonds) of the parental double helix by using ATP

Ligases seal nicks in DNA The energy is provided by ATP or NAD Phage--ATP E.coli--NAD * T 4 DNA ligases can join two blunt DS DNA 2- Enzymes/e: Ligase Eukaryote--ATP

D Dna A direct primosome to the origin na B( Helicase activity) na C & C’ are co-activator for primase na G( primase) S SSBP binds to each ss DNA, keep the separated strands apart T Tus recognize the ter sequence for termination of replication Protein Factors of Replication

Different Stages Recognition of the Ori sequence by Dna A Recognition of the Ori sequence by Dna A Initiation by primosome Initiation by primosome Elongation by replisome Elongation by replisome Termination at ter sequence by tus protein Termination at ter sequence by tus protein

Initiation of replication at Ori C

1- Recognition of the origin by Dna A molecules 2- Twisting of DNA around them result in a short unwinded DNA( loop) 3- Progressive unwinding of the DNA by Dna B and eye loop formation 4-Assembly of primosome at replication origin 5- Synthesis of the primer by primase followed by DNAP activity Recognition & Initiation of replication

Different ways of Priming reaction in replication 1-Primase: RNA polymerase Primer ~10 bases 2-Nicked DNA 3‘-OH 3-Terminal protein: Ser OH

2- Elongation: 5 ’ to 3 ’ PDE bond formation Replisome, a protein complex, associate with particular DNA structure to unwind the DNA and synthesis daughter strands. Pyrophosphate hydrolysis is necessary for irreversibility of this reaction

The two strands are run in opposite direction The DNA synthesis is only from 5 ’ to 3 ’ The two strands are synthesized Simultaneously But how ? One strand is made continuously (leading strand) and The other is made discontinuously (lagging strand, Okazaki fragments) Simultaneous synthesis of two strands of DNA

OKAZAKI FRAGMENT Its structure: A short sequence(10 nt) of RNA primer + about 100 nt of DNA How it is made: Primer by primase and DNA by DNAP III How it is joined to the other fragments: First the primer of the previous Okazaki fragment is hydrolyzed and replace by DNAP I, then the two ends of DNA is linked by ligase

Priming Extension Removal of primer Gap filling Nick ligation Discontinuous Replication Stages 1- Primer synthesis by primase 2- DNA synthesis by DNAP III 3- Removal of primer by DNAP I 4- DNA synthesis by DNAP I 5- Sealing the nick between two Okazaki fragments by ligase Nick translation: is referred to these last three stages

How the two strands are synthesize in same direction?

Synthesis of the two strands in same direction

3- Termination of repli- cation in E.Coli 1- Ter sequences is a consensus sequences with 23 bases 2- Tus protein is a 36 kDa protein that binds to the ter site 3- Contra helicase activity of the Ter- Tus complex 4- DNA replication stop 5- Separation of strand by topoisomerase

Replication of circular DNA in E. coli (3.10): 1.Two replication forks result in a theta-like () structure. 2.As strands separate, positive supercoils form elsewhere in the molecule. 3.Topoisomerases relieve tensions in the supercoils, allowing the DNA to continue to separate.

The fidelity of DNA replication Control at two different stages: 1- Presynthetic control: Control at the incoming base 2- Proofreading: Substitution Determined by 3 ’ to 5 ’ exonuclease activities

Replication in Eukaryotes The differences between Pro and Eu The Replication rate in eukaryotes could be low at least because: 1- DNA is so long 2- There are many physical barriers BUT How to overcome this problem? Having hundreds of origins

There are hundreds of Origin in eukaryotic genome, each is called ARS ARS (Autonomous replicating sequence)

The licensing factor in Yeast ORC: origin recognition complex, bind to A and B1 in ARS Cdc6, a highly unstable protein (half-life ＜ 5 min), synthesis only in the G1 phase Cdc6, allow Mcm bind to complex Replication initiation--Cdc6-Mcm are displaced

DNA polymerase δ replicase Priming replication repair location α βγε Nu Mito repair function replication Different DNAPs In Eukaryotic 5‘-3’ plolymerization 3‘-5’ exonuclease ’ -3‘exonuclease +----

Different Stages Recognition of the ARS sequence by ORC Recognition of the ARS sequence by ORC Initiation by DNAP α Initiation by DNAP α Elongation by replisome Elongation by replisome Termination by telomerase Termination by telomerase

Initiation of Replication in Eukaryotes

Elongation Nucleosome problem

End Replication Problem

Toxins and antibiotics that inhibit replication : makes cross link between the two strands of DNA, preventing them of being template Mitomycine : makes cross link between the two strands of DNA, preventing them of being template Nalidixic acid : Prevents gyrase activity Nalidixic acid : Prevents gyrase activity

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings. Fig Synthesis of telomeric DNA by telomerase