All nucleotides have a common structure

RNA DNA

Nucleoside = Adenine + riboseNucléotide = 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 CycleRegulators

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 wash & transfer

E. coli

N15 N14 N14

• E. coli first grown on a heavy isotope of Nitrogen (N15) in Generation 0 (G0), then bacteria washed and transferred to the lighter isotope N14 for both Generation 1 (G1) and Generation 2 (G2). Old DNA incorporated heavy isotope, while newly synthesized DNA must incorporate the light isotope.

G0 G1 G2

N15

N15 - yellow strand

N14 - black strand

N14

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’

5’

RNA primase RNA primase

RNA primer made; primase released

primase primase

DNA polymerase III extends DNA on RNA primerpol III pol III

5’3’ 5’

5’

5’3’

5’3’

5’3’

DNA polymerase III released

SIMPLIFIED STEPS IN DNA SYNTHESISSIMPLIFIED STEPS IN DNA SYNTHESIS

RP12

DNA template

RNA primer

DNA elongation

DNA polymerase I degrades RNA primer and fills in with DNA

pol I pol I

DNA polymerase I released

pol I pol I

DNA ligase facilitates covalent closure of final two nucleotides (black)

ligase ligase

ligase released, new strand completed

ligase ligase

5’5’3’3’

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 Single-stranded template Complementary base-pairing

(fig10.4a)

DNA Synthesis Occurs in the 5’3’ Direction

P P P P P PP PP

PP PP5’

3’ 5’

OH 3’

OH 3’

P P P P P PP PP

PP PP5’

3’ 5’

OH3’5’PPP

P P P P P PP PP

5’

3’ 5’

OH 3’PP PP P

PP

Incoming nuceolotidetriphosphate

Nucleotide monophosphateadded to chain with release

of diphosphate

DNA SynthesisDNA 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

Enzyme Primary function

DNA Pol I (PolA) Major DNA repair enzymeDNA Pol II DNA repairDNA Pol III De novo synthesis of new DNA

_______________________________________________

MAMMALIAN

Enzyme Primary function Location

DNA Pol I () Strand synthesis initiation NucleusDNA Pol II () DNA repair NucleusDNA Pol III () Strand extension NucleusDNA Pol DNA repair NucleusDNA Pol De novo synthesis of new DNA Mitochon.

Arthur Kornberg - Nobel Prize for isolating DNA polymerase I

Properties I II III

Initiate Chain Synthesis _ _ _

5’ to 3’ polymerization + + +

3’ to 5’ exonuclease activity + + +

5’ to 3’ exonuclease activity + _ _

Prokaryotic DNA Polymerases

RP7

DNA Polymerase IDNA Polymerase I

This is the best understood of the DNA polymerases

5’3’ exo 3’5’ exo PolymeraseN- -C

36 kD 67 kDKlenow Fragment of DNA Pol I(Used widely in labs since it avoids DNA degradation mediated by 5’ exo)

proteolytic cleavageyields the ~67 kDaKlenow 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

BACTERIAL CHROMOSOME

EUCARYOTIC CHROMOSOME

ori ori 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 proteins9-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 DNAligasepresent

T4 DNA ligase absent Phage T4 DNAs were

labelled with very short pulsesseparated accordingto size byultracentrifugation

Absence of DNAligase leads to theaccumulation of veryshort pieces of DNA

OkazakiDNAfragments

•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 Looping of template for the lagging strand enables a dimeric DNA polymerase III holoenzyme at the replication fork to DNA polymerase III holoenzyme at the replication fork to

synthesize both of the daughter strandssynthesize 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’ PO4 and 3’ OH• Ends must be physically close• Energy requiring reaction

18

Mutation, mutants & mutagensMutation • 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 mutationMutations at the DNA level1. Point mutationThis 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 deletionThe addition or removal of one or more base-pairs.

Mismatches can cause mutationswhen 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 105 - 106 bases during replication• In DNA, 1 mistake every 108 - 109 bases

Proofreading by DNA polymerase III

(3’ -> 5’ exonuclease activity)