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DNA replication
Learning objectivesUnderstand the basic rules
governing DNA replicationIntroduce proteins that are typically
involved in generalised replication
Reference: Any of the recommended texts
Optional Nature (2003) vol 421,pp431-435http://www.bath.ac.uk/bio-sci/cbt/
http://www.dnai.org/lesson/go/2166/1973
`It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material’
Watson & Crick Nature (1953)
Original drawing by Francis Crick
Four requirements for DNA to be genetic material
Must carry information Cracking the genetic code
Must replicate DNA replication
Must allow for information to change Mutation
Must govern the expression of the phenotype Gene function
Much of DNA’s sequence-specific information is accessible only when the double helix is unwound
Proteins read the DNA sequence of nucleotides as the DNA helix unwinds. Proteins can either bind to a DNA sequence, or initiate the copying of it.
Some genetic information is accessible even in intact, double-stranded DNA molecules
Some proteins recognize the base sequence of DNA without unwinding it.
One example is a restriction enzyme.
DNA stores information in the sequence of its bases
DNA Replication
Process of duplication of the entire genome prior to cell division
Biological significance
extreme accuracy of DNA replication is
necessary in order to preserve the integrity
of the genome in successive generations
In eukaryotes , replication only occurs during
the S phase of the cell cycle. The slower
replication rate in eukaryotes results in a
higher fidelity or accuracy of replication in
eukaryotes
Basic rules of replication
A. Semi-conservativeB. Starts at the ‘origin’C. Can be uni or bidirectionalD. Semi-discontinuousE. Involves DNA templatingF. RNA primers required
A) DNA replication – three possible models
Semiconservative replication – Watson and Crick model
Conservative replication: The parental double helix remains intact; both strands of the daughter double helix are newly synthesized
Dispersive replication: At completion, both strands of both double helices contain both original and newly synthesized material.
Meselson-Stahl experiments confirm semiconservative replication
Semi-conservative replication An ingenious experiment by Meselson and Stahl showed that one strand of each DNA strand is passed on unchanged to each of the daughter cells. This 'conserved' strand acts as a template for the synthesis of a new, complementary strand by the enzyme DNA polymerase
Meselson-Stahl experiments confirm semiconservative replication
Double helix unwinds
Each strand acts as template
Complementary base pairing
Two daughter helices produced after replication
Starts at origin
Initiator proteins identify specific base sequences on DNAProkaryotes – single ori site E.g E.coli - oriC
Eukaryotes – multiple sites of origin (replicator)E.g. yeast - ARS (autonomously replicating sequences)
ProkaryotesEukaryotes
Uni or bidirectional
Bidirectional replication Replication forks move in opposite
directions In linear chromosomes, telomeres ensure
the maintenance and accurate replication of chromosome ends
In circular chromosomes, such as E. coli, there is only one origin of replication.
In circular chromosomes, unwinding and replication causes supercoiling, which may impede replication
Topoisomerase – enzyme that relaxes supercoils by nicking strands
The bidirectional replication of a circular chromosome
Semi-discontinuous replicationAnti parallel strands replicated simultaneously Leading strand synthesis continuously in 5’– 3’ Lagging strand synthesis in fragments in 5’-3’
Semi-discontinuous replication
New strand synthesis always in the 5’-3’ direction
DNA templating
Nucleotide recognitionEnzyme catalysed polymerisation
Complementary base pair copiedSubstrate used is dNTP
RNA primers required
TopoisomerasesHelicases
Single strand binding proteins
PrimaseDNA polymerase
Tethering protein
DNA ligase
- Prevents torsion by DNA breaks
- separates 2 strands - prevent reannealing of single strands- RNA primer synthesis- synthesis of new strand- stabilises polymerase - seals nick via
phosphodiester linkage
Core proteins at the replication fork
The mechanism of DNA replication
Arthur Kornberg, a Nobel prize winner and other biochemists deduced steps of replication Initiation
Proteins bind to DNA and open up double helix Prepare DNA for complementary base pairing
Elongation Proteins connect the correct sequences of
nucleotides into a continuous new strand of DNA
Termination Proteins release the replication complex
Core proteins at the replication fork
Nature (2003) vol 421,pp431-435 Figure in ‘Big’ Alberts too
Topoisomerase I
Cause temporary single strand breaks in DNA Allows free rotation of helixMonomeric enzymes (~100kD)Reversible catenation (knotting)Acts by breaking phosphodiester linkage via tyrosine active site
Topoisomerase II (DNA gyrase)
Cause temporary double strand breaks in DNA
Topoisomerase II (DNA gyrase)
~ 375 kD2 pairs of subunits: A & BATP hydrolysis mediated double strand breakCatenates double stranded circlesInhibited by antibiotics