replication of nucleic acids. 2 because sometimes this... 3
TRANSCRIPT
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Replication of Nucleic Acids
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Because sometimes this...
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...leads to this! (Yikes!)
http://www.scienceclarified.com/Ex-Ga/Fertilization.html4
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Cell Numbers must increase to get growth of an organism Cells duplicate their
contents One parental cell
gives rise to two daughter cells (blue)
Duplication of DNA must occur
Mutation rate must be low – replication with high fidelity
Mutation rate is 1 nucleotide/109 nucleotides (6.4x109 bp in a human diploïd cell)
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DNA replication in cell cycle The cell cycle is the
ordered process of cellular duplicationThe replication of
DNA only occurs in the S phase of the cell cycle
M phase: mitosisG1 and G2: gaps, the
cell gets ready for the next phase
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Distribution of DNA strands – the possibilities There are different
possibilities:1 parental:1 daughter
strand (semi-conservative) (Watson-Crick)
Parental and daughter (conservative) (Bloch)
Breaks in DNA (dispersive) (Delbrück)
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http://web.virginia.edu/Heidi/chapter30/chp30.htm
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The Meselson-Stahl experiment – the setup 14N (light) is the
most abundant form of nitrogen
15N (heavy) is not radioactive, but heavier
Centrifuge 1:1 mixture of DNA in CsCl gradient, then take the UV absorption reading
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Meselson M, Stahl FW. 1958. The replication of DNA in Escherichia coli. PNAS Vol 44: 671-682
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The Meselson-Stahl experiment – the results Generations are
measured from time added 14N
At generation 0: one band
At generation 1: 1 band
At generation 1.9: 2 bands
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Why are there two bands at generation 1.9?Why are there three bands
when you mix 0 and 1.9?
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Distribution of DNA strands
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This one is proven!
http://upload.wikimedia.org/wikipedia/commons/a/a2/DNAreplicationModes.png
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Basic needs for DNA replicationIn order to start, the following are required: All of the 4 deoxyribonucleotide
triphosphates (dNTPs) dATP, dTTP, dCTP, dGTP
DNA template DNA/RNA primer to start the replication DNA polymerase
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DNA replication Base pairing occurs
according to Watson-Crick rules
Hydrolysis gives rise to a phosphodiester bond
Releases pyrophosphate (favourable energy release)
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Phosphodiester bond formation
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Nature. 1998. 391:231-232
The incoming nucleotide has 2 metal ions (Mg2+) attached to the phosphates
The Mg2+ also binds to Asp residues (conserved)
Lowers the affinity of O for H on 3’ OH, and allows for the reaction to occur
The exposed –OH group reacts with the triphosphate group of the incoming base
3’ 5’5’ 3’
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DNA synthesis - polymerase
The incoming nucleotide must pair with the template strand to be recognized by the polymerase
DNA polymerase is the catalyst to the formation of the phosphodiester
Processivity is the number of phosphodiester bonds formed by the polymerase before it falls off the DNA
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DNA Replication
18Figure 5-6 Molecular Biology of the Cell (© Garland Science 2008)
On replicating bacterial DNA, two Y-shaped structures are occurring at the same time
Each arm of the Y: both replicated strands
The start point of replication is called the replication origin
Large DNA molecules can have many origins of replication
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DNA replication fork DNA is denatured (unwound) Replication always occurs 5’→3’ in living organisms An experiment with 3H in bacteria showed the
presence of DNA strands 1000-2000nt long: the Okazaki fragments
Okazaki in eukaryotes: 100-200nt long
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Primers for the lagging strand Primers are needed
every 100-200 nucleotides on lagging strand
Primers are made by the DNA primase
Made with RNA About 10nt length
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Replicating the lagging strand RNA primers are extended by
DNA polymerase III Polymerase falls off when
encounters double-strand structure
DNA polymerase I system removes RNA and replaces DNA
DNA ligase forms diester bonds
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Types of DNA polymerases
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Keeping the polymerase on Polymerase will not
stay on very long on its own
The sliding clamp keeps the polymerase on the leading strand
Clamp is loaded at the primer/template junction by the clamp loader
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ATP-dependant!
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Polymerase – more than a protein...
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The sum of all parts
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Each shape is a protein. Together, they form a complex that makes up the polymerase
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Proofreading DNA polymerase III
has 3’→5’ exonuclease activity
A mismatched pairing causes the transfer of DNA strand from the polymerizing subunit to the editing subunit
The mismatched nucleotide is removed
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Proofreading...
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DNA Ligase
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Phosphodiester bond is missing on the DNA strand
In bacteria: ligase is NAD+ dependentIn eukaryotes: is ATP dependant
DNA ligase residue
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Unwinding DNA To be replicated,
DNA must be opened in front of the fork
Double helix very stable
Helicase uses ATP to propel it along the strand
Opens DNA helix at up to 1000bp/s
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Keeping it straight Hairpins in ssDNA
will destabilize the polymerase
Single-strand DNA-binding protein (SSB) bind tightly and destabilize the helix
SSB do not cover the bases
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The origin
In E. coli: Major initiator
protein is DnaAThe region is near
an AT rich region
Helicase is DnaB Primase is DnaG
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The active replication fork
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Putting it all together
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DNA sequencing – Sanger Method
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Each ddNTP is labelled with a different fluorescent dye
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PCR Standard technique in
medical and research labs
After 25 cycles, the target sequence is amplified in the order of 106 (2n =n is number of cycles)
Used to identify pathogens in infections, cancer types, genetic disorders...
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https://sites.google.com/a/luther.edu/genetics/students/tyler-best/pcr-amplification