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