DNA ReplicationDNA ReplicationSection 4.3Page 217

Why do we need to replicate our DNA?When does DNA replication occur in a cell?

BackgroundBackgroundCell division: mitosis + cytokinesis

DNA replicated in interphase, prior to mitosis

Each daughter cell must have an exact copy of the parent cell’s DNA

But how does replication occur? But how does replication occur? Scientists in the 50s had 3 proposed Scientists in the 50s had 3 proposed models:models:

1. Semi-conservative2. Conservative3. Dispersive

But which one is right???

Semi-conservative – Two parental strands separate and each serves as a template for a new progeny strand.

Newly-synthesized DNA molecules has one old strand,and one new strand

Conservative – the two parental strands stay together, and somehow produce another daughter helix with completely new strands.

Newly-synthesized DNA molecules has two new strands.

Dispersive – DNA becomes fragmented so that new and old DNA coexist in the same strand after replication.

Newly-synthesized DNA molecule has pieces of oldand new strands interspersed.

Meselson-Stahl experiment, 1958Meselson-Stahl experiment, 1958

Purpose: to elucidate the mode of replication

Experimental model: E. coli

Grew E. coli in a medium enriched with a heavy nitrogen isotope (15N)◦Denser-than-normal DNA

Switched cells to ordinary medium with 14N, and allowed DNA replication

Will 14N be incorporated into DNA strands with 15N?

Centrifuged the DNA within a density gradient: Separates components according to density

*A centrifuge is a device that spins a solution at high speeds, the spinning splits up the different components in a mixture based on density

http://highered.mcgraw-hill.com/olc/dl/120076/bio22.swf

Possible results:

Observed:First generation – One intermediate bandSecond generation – One light/one

intermediate

Conclusion: DNA replication is semi-conservative

DNA REPLICATION:DNA REPLICATION:THE DETAILSTHE DETAILS

Three stages:Three stages:Stage 1: Initiation◦DNA strands are separated◦A small portion of RNA is annealed to the

exposed strands to “prime” them for replication

Stage 2: Elongation:◦ DNA polymerase III builds a new strand of DNA

by incorporating nucleotides

Stage 3: Termination

Stage 1: InitiationStage 1: Initiation

Separation of strands:

DNA strands are “unzipped” by DNA helicase◦Hydrogen bonds between complementary bases

are broken

Single-stranded binding proteins (SSBs) bind to exposed strands to prevent re-annealing of strands.

DNA gyrase relieves torsion tension by cutting and re-annealing the two strands

Priming:

DNA polymerase cannot start incorporating nucleotides on its own◦Needs an existing 3’ end of a nucleic acid

A short segment of RNA (a “primer” – 10 to 60 nucleotides long) provides that 3’ end

RNA Primase synthesizes the primer and anneals it to the template strand.

DNA polymerase can then add on DNA nucleotides

Stage 2: ElongationStage 2: ElongationNew strand is synthesized in the 5’ to 3’ direction

(added on to the end with the -OH group)

Catalyzed by DNA polymerase III

Free bases are floating in the nucleoplasm as deoxyribonucleoside triphosphates.

Energy required for DNA synthesis is provided by hydrolyzing the bond between the 1st and 2nd phosphates

Characteristics of elongation:Characteristics of elongation:

A. Bi-directionalityB. Semi-discontinuity

A. Elongation is bi-directional

Elongation proceeds in two directions, outwards from the origin of replication.

The junction where the strands are still joined is called the replication fork.

DNA synthesis occurs simultaneously using both strands as templates◦ A replication bubble forms between two replication

forks

B. Elongation is semi-discontinuous

DNA synthesis always occurs in the 5’ to 3’ direction (of the new strand!)

The two template strands are antiparallel Only one strand can be built continuously

http://highered.mcgraw-hill.com/olc/dl/120076/micro04.swf

Leading strand – Uses the 3’ to 5’ template strand as its guide◦ Is built continuously, towards the replication fork

Lagging strand – Uses the 5’ to 3’ template strand as its guide◦ Is built discontinuously in short fragments

RNA primase constantly adds new RNA primers along the template strand.

The fragments are called Okazaki fragments.

= site of new primer

Removal of the RNA primers, and joining of the Okazaki fragments:

Enzyme Role

DNA polymerase I removes the RNA primers;

replaces them with the proper deoxyribonucleosides

DNA ligase joins the fragments together (phosphodiester bonds)

Removal of the RNA primers, and joining of the Okazaki fragments:

Stage 3: TerminationStage 3: TerminationTwo replication forks meet each other; orDNA Polymerase III reaches the end of a

strand

Problem: Shortening of telomeres

Telomeres: The ends of DNA. Contain repetitive sequences.

Protects the chromosome from degradation.

Loss of telomeric DNA occurs on the lagging strand with each replication.

http://spine.rutgers.edu/cellbio/assets/flash/tel.htm

Lagging strand:

No free 3’ end to replace RNA with DNA

Approximately 50 replications before the telomeres become too short.

Telomere shortening linked to aging.

TelomeraseTelomeraseTelomerase - enzyme that prevents shortening

of telomeres

Present in cells that need to divide constantly: white blood cells, germ line cells

May be present in cancerous cells

ProofreadingProofreadingDNA polymerase III and DNA polymerase I

are constantly proofreading the progeny strand as it is synthesized.

Both have exonuclease activity can identify incorrectly added nucleotides, backtrack,

and excise them (cut them out) before continuing synthesis.

Further proofreading mechanisms are in place when synthesis is completed.

Recap:Recap:

Three important properties of DNA replication:

1.DNA replication is semi-conservative2.DNA replication is bi-directional3.DNA replication is semi-discontinuous

…recall what these mean!!

Enzymes/proteins involved in DNA replication:

DNA helicaseDNA gyrasesingle-stranded binding proteinsRNA primaseDNA polymerase IIIDNA polymerase IDNA ligaseDNA telomerase