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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
Cell CycleRegulators
Replication Commitment
Cell Growth & Completion of
Replication
Cell Division
Cell Division and DNA Replication (procaryotes)
Replication Initiation
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
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
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 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!)
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
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
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
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
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