All nucleotides have a common structure RNADNA Nucleoside = Adenine + ribose Nucléotide = Adenine + ribose + phosphate

Nucleotide subunits are linked together by phosphodiester bonds 3’ R-O: 3’OH = nucleophilic chatacter 5’P = electrophilic character

BASE PAIRING AND HYDROGEN BOUNDING

Cell Cycle Regulators Replication Commitment Cell Growth & Completion of Replication Cell Division Cell Division and DNA Replication (procaryotes) Replication Initiation

Bacterial replication: a new round is initiated before the first round is complete

If humans did not have multiple origins of replication, then replication of the genome from a single origin with two forks would take several weeks

Nature (1953), 171:737 “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” DNA Replication

Alternative Models for DNA Replication Semi-conservative Conservative Dispersive Semi-conservative - Old strand conserved, new strand synthesized off of old. Conservative - Both old strands conserved in double helix, two new strands interpreted from old Dispersive - Old strands break into pieces, new DNA synthesized and reorganized into mixture of old and new pieces of DNA RP2

Matthew Messelson Franklin Stahl SEMI CONSERVATIVE DNA REPLICATION (1958)

Meselson and Stahl : Demonstration of Semi- conservative Replication wash & transfer E. coli N 15 N 14 E. coli first grown on a heavy isotope of Nitrogen (N 15 ) in Generation 0 (G0), then bacteria washed and transferred to the lighter isotope N 14 for both Generation 1 (G1) and Generation 2 (G2). Old DNA incorporated heavy isotope, while newly synthesized DNA must incorporate the light isotope. G0G1 G2

N 15 N 15 - yellow strand N 14 - black strand N 14 G0 G1 G2 heavy intermediate light Proof DNA Replication Semi-conservative DNA centrifuged in Cesium Chloride, heavy DNA settles lower in tube

DNA REPLICATION : THE OVERALL PROCESS

3’ 5’ RNA primase RNA primer made; primase released primase DNA polymerase III extends DNA on RNA primer pol III 5’ 3’5’ 3’ 5’ 3’ 5’ 3’ DNA polymerase III released SIMPLIFIED STEPS IN DNA SYNTHESIS RP12 DNA template RNA primer DNA elongation

DNA polymerase I degrades RNA primer and fills in with DNA pol I DNA polymerase I released pol I DNA ligase facilitates covalent closure of final two nucleotides (black) ligase ligase released, new strand completed ligase 5’ 3’ Rnase activity DNA

DNA Replication (2) DNA replication requires assembly of many proteins (at least 30) at a growing replication fork: helicase to unwind primase to prime polymerase to elongate the chain ligase to ligate (join) topisomerases to remove supercoils DNA polymerases are enzymes that copy (replicate) DNA DNA polymerases require a short preexisting DNA strand (primer) to begin chain growth. DNA polymerase adds nucleotides to the free hydroxyl group at the 3’ end of the primer.

DNA REPLICATION : THE CHEMICAL ASPECT

DNA Replication n Single-stranded template n Complementary base-pairing (fig10.4a)

DNA Synthesis 2 phosphates - nucleotide gets positioned through H- bonding with template - 3’-OH nucleophilic attack on alpha phosphate of incoming dNTP. - loss of entropy; not much gain in bond-energy - reaction is driven by removal and splitting of pyrophosphate - because of requirement for 3’-OH and 5’ dNTP substrate, reaction only occurs in the 5’  3’ direction (direction of new strand!)

DNA POLYMERASES ARE DOING THE JOB

The Major DNA Polymerases BACTERIAL EnzymePrimary function DNA Pol I (PolA)Major DNA repair enzyme DNA Pol IIDNA repair DNA Pol IIIDe novo synthesis of new DNA _______________________________________________ MAMMALIAN EnzymePrimary functionLocation DNA Pol I (  )Strand synthesis initiationNucleus DNA Pol II (  )DNA repairNucleus DNA Pol III (  )Strand extensionNucleus DNA Pol  DNA repairNucleus DNA Pol  De novo synthesis of new DNAMitochon.

Arthur Kornberg - Nobel Prize for isolating DNA polymerase I Properties I II III Initiate Chain Synthesis _ _ _ 5’ to 3’ polymerization ’ to 5’ exonuclease activity ’ to 3’ exonuclease activity + _ _ Prokaryotic DNA Polymerases RP7

DNA Polymerase I This is the best understood of the DNA polymerases 5’  3’ exo 3’  5’ exo Polymerase N- -C 36 kD 67 kD Klenow Fragment of DNA Pol I (Used widely in labs since it avoids DNA degradation mediated by 5’ exo) proteolytic cleavage yields the ~67 kDa Klenow Fragment - 3’ exonuclease degrades single-stranded DNA from 3’ end - 5’ exonuclease degrades base paired DNA from the 5’ terminus -polymerase adds nucleotides

Tertiary structure of Klenow fragment of DNA polymerase I (has catalytic and proofreading (3’ to 5’ exonuclease) activity Protein structure: Alpha helices (barrels), Beta sheets (flat arrows) and loops

THE ORIGIN OF REPLICATION

Procaryotic (Bacterial) and Eucaryotic Chromosome Replication ori ter B ACTERIAL C HROMOSOME E UCARYOTIC C HROMOSOME ori Replication occurs at a specific site on the DNA called the replication origin. Replication initiation proteins bind to the DNA and pry the two strands apart. The replication origin occurs at a site where the DNA helix is easier to pull apart: A-T base pairs. Bacterial genome has a single origin of replication while the humangenome has ~10,000

Origin of Replication Replication has defined start site Sequence recognized by “initiator protein” Prokaryotes have one on circular chromosome Eukaryotes have many per linear chromosome 10 Sites for DNA binding proteins 9-mer sequences

Initiation of replication at oriC DnaA binds and begins to melt double helix Helicase (DnaB) continues to separate strands

SYNTHESIS DIRECTION AND OKASAKI FRAGMENTS

Semidiscontinuous DNA synthesis is 5’ to 3’ However double helix is antiparallel Replication is continuous on one strand (leading) and discontinuous on other strand (lagging)

Experimental demonstration of Okazaki fragments using pulse labelling and size fractionation by utracentrifugation T4 DNA ligase present T4 DNA ligase absent Phage T4 DNAs were labelled with very short pulses separated according to size by ultracentrifugation Absence of DNA ligase leads to the accumulation of very short pieces of DNA Okazaki DNA fragments

only the leading strand can be replicated in a continuous fashion. The DNA being synthesized on the lagging strand must be made as a series of short fragments (Okazaki fragments) that will be joined together at a later time. The pieces are stitched together using a DNA ligase enzyme to form a continuous new strand.

Looping of template for the lagging strand enables a dimeric DNA polymerase III holoenzyme at the replication fork to synthesize both of the daughter strands

Replication fork- complex view Single-strand DNA binding protein Sliding clamp

DNA LIGATION AND TERMINATION

DNA Ligase Joins DNA ends together (not add bases onto strand!) Forms bond between 5’ PO 4 and 3’ OH Ends must be physically close Energy requiring reaction 18

Mutation, mutants & mutagens Mutation a change in the base sequence of DNA (generally this is with in a gene). these changes can include base substitution, addition, re-arrangement or deletion (& multiples thereof). Mutant an organism carrying a mutation. by implication it should have a mutation in a gene which makes it distinct from normal (Wild-Type). Mutagen a physical or chemical agent that causes a mutation.

Types of mutation Mutations at the DNA level 1. Point mutation This is the replacement of a single nucleotide for another, i.e., change of base. 2 types: Transition – a change of purine to purine (A to G, G to A) or pyrimidine to pyrimidine (C to T, T to C) Transversion – a change of purine to pyrimidine or vicer-versa, e.g. A to C or T, C to A or G 2. Insertion or deletion The addition or removal of one or more base-pairs.

Mismatches can cause mutations when the DNA is replicated 5’-ATTGG-3’ 3’-TAACC-5’ 5’-ATGGG-3’ 3’-TAACC-5’ 5’-ATGGG-3’ 3’-TACCC-5’ 5’-ATTGG-3’ 3’-TAACC-5’ Normal Mutated Replication 1 mistake every bases during replication In DNA, 1 mistake every bases

Proofreading by DNA polymerase III (3’ -> 5’ exonuclease activity)