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

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

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

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?

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’

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

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!

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

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

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

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