DNA REPLICATION

Anna Onofri

Liceo «I.Versari»

Learning objectives

1. Understand the basic rules governing DNA replication

2. Understand the function of key proteins involved in a generalised replication model

“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.

DNA stores information in the sequence of its bases

Some proteins recognize the base

sequence of DNA without unwinding it

(e.g. a restriction enzyme).

restriction enzyme EcoR V

DNA replication occurs with great fidelity

Somatic cell DNA stability and reproductive-cell DNA

stability are essential. Why?

Identity

Genetic diseases

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.

• Replication rate in eukaryotes is slower resulting in a

higher fidelity/accuracy of replication in eukaryotes

The mechanism of DNA replication

Arthur Kornberg et al (Nobel 1959)

He was the first to discover how molecules of deoxyribonucleic acid (DNA) duplicate within bacterial cells and also the first to devise a method for synthesizing this process in a laboratory setting.

For these achievements he shared the 1959 Nobel Prize in physiology or medicine with Severo Ochoa.

REPLICATION STEPS• 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

Basic rules of replication

A. Semi-conservative

B. Starts at the ‘origin’

C. Can be uni or bidirectional

D. Semi-discontinuous

E. Synthesis always in the 5-3’ direction

F. RNA primers required

DNA replication Of the 3 possible models, replication is…

A) Semi-

conservative

Meselson and Stahl experiment

• Meselson and Stahl concluded that DNA employs semi-conservative replication. They placed the bacteria in an environment which contained a selected Nitrogen isotope.

• They first used N14. The bacteria then integrated this isotope into their DNA. Later they used an environment that contained N15.

• They then looked at which of the isotopes the bacterial DNA contained.

• They found that it contained both isotopes of nitrogen, which implies that conservative replication is not the correct conclusion

Semiconservative replication

Half the original DNA molecule is

saved, or conserved in the daughter

molecules. This is why the process

is called semi-conservative.

B) Starts at originInitiator proteins identify specific base sequences on DNA called sites of origin

Prokaryotes – single origin site E.g E.coli - oriC

Eukaryotes – multiple sites of origin (replicator)

E.g. yeast - ARS (autonomously replicating sequences)

Prokaryotes Eukaryotes

C) Uni or bidirectionalReplication forks move in one or opposite directions

D) Semi-discontinuous replication

Anti 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

E) Synthesis is ALWAYS in the 5’-3’ direction

Nucleotide recognition

Enzyme catalysed polymerisation (DNA polymerase)

Complementary base pair copied

Substrate used is dNTP

Where does energy for addition

of nucleotide come from?

What happens if a base

mismatch occurs?

DNA polymerase has 3’ 5’ exonuclease activity in order to correct errors

From cleavage of high energy phosphate of incoming triphosphate

PROOFREADING

• The term proofreading is used in genetics to refer to the error-correcting processes,.

• When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA and excises the mismatched base. Following base excision, the polymerase can re-insert the correct base and replication can continue.

F) RNA primers required• DNA polymerase can only join an incoming nucleotide to one that is base-paired

• RNA primase provides a base paired 3’ end as a starting point for DNA pol by synthesising ~10 nucleotide primers

Core proteins at the replication fork

- regulate the overwinding or underwinding of DNA

- separates 2 strands

- RNA primer synthesis

- synthesis of new strand

- seals nick via phosphodiester linkage

Topoisomerase

Helicases

Primase

DNA polymerase

DNA ligase