What Is DNA Replication

• DNA Replication is the process in which the DNA within a cell makes an exact copy of itself.– Why does DNA replicate?

– During which phase of the cell cycle does DNA replicate?

Outline for ReplicationReplication

I. General Features of ReplicationA. Semi-ConservativeB. Starts at OriginC. BidirectionalD. Semi-Discontinuous

II. Proteins and Enzymes of Replication

III. Detailed Examination of the Mechanism of ReplicationA. InitiationB. PrimingC. ElongationD. Proofreading and Termination

Identical base sequences

5’

5’

3’

3’ 5’

5’3’

3’

Watson/Crick proposed mechanism of DNA replication

• In the late 1950s, three different mechanisms were proposed for the replication of DNA– Conservative model

• Both parental strands stay together after DNA replication

– Semi conservative model• The double-stranded DNA contains one parental and one

daughter strand following replication

– Dispersive model• Parental and daughter DNA are interspersed in both strands

following replication (DNA broken every 10 bp to prime short regions that are joined later).

Proposed Models of DNA ReplicationProposed Models of DNA Replication

Alternative models of DNA replication

Evidence for strand separation during replication

• Matthew Meselson and Franklin Stahl experiment in 1958

“The most beautiful expt in biology”– Grow E. coli in the presence of 15N (a heavy isotope of Nitrogen)

for many generations• Cells get heavy-labeled DNA

– Switch to medium containing only 14N (a light isotope of Nitrogen)– Collect sample of cells after various times– Analyze the density of the DNA by centrifugation using a CsCl

gradient

CsClCsCl equilibrium equilibrium Density Gradient CentrifugationDensity Gradient Centrifugation

= 1.65

= 1.8

DNA bands where its density is equal to that of CsCl

1958: Matthew Meselson

& Frank Stahl’s Experiment

Semiconservative

model of DNA replication

Interpreting the Data

After one generation, DNA is “half-heavy”

After ~ two generations, DNA is of two types: “light”

and “half-heavy”

This is consistent with only the semi-conservative model

1955: Arthur Kornberg

Worked with E. coli (used cell-free extracts for test-tube synthesis of DNADiscovered the mechanisms of DNA synthesis.

Four components are required:

1.

dNTPs: dATP, dTTP, dGTP, dCTP(deoxyribonucleoside

5’-triphosphates)(sugar-base + 3 phosphates)

2.

DNA template

3.

DNA polymerase I (Kornberg enzyme)

4.

Mg 2+ (optimizes DNA polymerase activity)

No other non-DNA intermediates formed in this cell-free process

1959: Arthur Kornberg (Stanford University) & Severo Ochoa (NYU)

Three main features of the DNA synthesis reaction:

1.

DNA polymerase I catalyzes formation of phosphodiester

bondbetween 3’-OH of the deoxyribose

(on the last nucleotide) and the 5’-phosphate of the dNTP.

• Energy for this reaction is derived from the release of two of the three phosphates.

2.

DNA polymerase “finds”

the correct complementary dNTP

at each step in the lengthening process.

• rate ≤

800 dNTPs/second• low error rate

3.

Direction of synthesis is 5’

to 3’

DNA polymeraseImage credit:Protein Data Bank

DNA elongation

DNA elongation

Pyrophosphate

Pyrophosphate

waterPhosphateG = -6 kcal/mol

Outline for ReplicationReplication

I. General Features of ReplicationA. Semi-Conservative B. Starts at OriginC. BidirectionalD. Semi-Discontinuous

II. Proteins and Enzymes of Replication

III. Detailed Examination of the Mechanism of ReplicationA. InitiationB. PrimingC. ElongationD. Proofreading and Termination

Origin of replication (e.g., the prokaryote example):

Begins with double-helix denaturing into single-strands thus exposing the bases.

Exposes a replication bubble

from which replication proceeds in both directions.

~245 bp in E. coli

• DNA synthesis begins at a site termed the origin of replicationorigin of replication

• Each bacterial chromosome has only one

BACTERIAL REPLICATION BACTERIAL REPLICATION

• The origin of replication in E. coli is termed oriC– origin of Chromosomal replication

• Important DNA sequences in oriC–– ATAT--rich regionrich region–– DnaADnaA boxesboxes

Initiation of Replication

Figure 11.5 DNA sequences at the Bacterial origin of ReplicationFigure 11.5 DNA sequences at the Bacterial origin of Replication

Synthesis of DNA proceeds bidirectionallybidirectionally around the bacterial chromosome

eventually meeting at the opposite side of the bacterial chromosome

Where replication ends

BACTERIAL REPLICATION BACTERIAL REPLICATION

Overview of bacterial DNA replicationOverview of bacterial DNA replication

John CairnsVisualised E. coli chromosomesTheta configuration of replicating E. coli chromosome

Replication Starts at an Replication Starts at an OriginOrigin and is and is BidirectionalBidirectional

Directionality of the DNA strands at a replication forkDirectionality of the DNA strands at a replication fork

Leading strand

Lagging strandFork movementFork movement

DNA Replication

Replicationbubble

Replicationfork

Replicationfork

Hydrogen bond

DNA replication is the process where an entire double-stranded DNA is copied to produce a second, identical DNA double helix.

DNA Replication

• Helicase unwinds the double helix starting at a replication bubble.

• The two strands separate as the hydrogen bonds between base pairs are broken.

• Two replication forks form and the DNA is unwound in opposite directions.

DNAhelicase

DNA Replication

•Helicase has completed unwinding the DNA strand.

•Single strand Binding Proteins (SSB) keep the two strands from re-annealing (coming back together).

DNA Replication

Primase RNA Primer

•Primase is an RNA polymerase that makes the RNA primer.

•These primers “tell” the DNA polymerase where to start copying the DNA.

Leading Strand

Lagging Strand

DNA Replication

DNA Polymerase

• The DNA polymerase starts at the 3’ end of the RNA primer of the leading stand CONTINUOUSLY.• DNA is copied in 5’ to 3’ direction.• DNA polymerase copies the lagging strand DIS- continuously.

Leading Strand

Lagging Strand

3’ 5’

5’ 3’

Direction of Replication

Direction of Replication

DNA Replication

• The dis-continuous pieces of DNA copied on the lagging strand are known as Okazaki fragments.

DNA Replication

Another DNA Polymerase removes the RNA primers and replaces them with DNA.

DNA Replication

Finally the gaps in the sugar phosphate backbone are sealed by DNA ligase

There are now 2 identical double helices of DNA.

ligase

Outline for ReplicationReplication

I. General Features of ReplicationA. Semi-Conservative B. Starts at OriginC. BidirectionalD. Semi-Discontinuous

II. Proteins and Enzymes of Replication

III. Detailed Examination of the Mechanism of ReplicationA. InitiationB. PrimingC. ElongationD. Proofreading and Termination

Possible modes of DNA replication

Reiji Okazaki

Experimental demonstration of semisemi--discontinuousdiscontinuous Replication3H labeled Okazaki fragments seen in sucrose density gradients

Wild-type cells DNA Ligase deficient cells

Sucrose gradient

Top=Smaller

Bottom=Bigger

Long DNA piecesShort DNA pieces