DNA replication. SBI4U0- DNA Replication Cells need to have the ability to reproduce for many reasons. When cells make copies of themselves, they must.

DNA replication

SBI4U0- DNA Replication Cells need to have the ability to reproduce for

many reasons. When cells make copies of themselves, they

must ensure that their entire genetic code is present in both the old and new cell

DNA must replicate to ensure 2 complete copies of the genetic code. This is why cells must undergo the process of MITOSIS (remember, sister chromatids are copies of the same chromosome!)

DNA Replication

During cell division, the genetic material has to be divided up between daughter cells or gametes. (Mitosis - production of daughter cells; Meiosis- production of gametes)

DNA replicates first in INTERPHASE

The Cell Cycle

Remember This?

The Cell Cycle

Two Main Stages1.Interphase Growth 1, Synthesis, Growth 22.Cell Division Mitosis, Cytokinesis

THE CELL CYCLE

Prophase

MetaphaseAnaphase

Telophase

Cytokinesis

G1

S

G2

INTERPHASE

Period between cell divisionsCell undergoes growth, replication of DNA, obtaining energy, making hormones, repairing damage, fighting disease

Prophase

Metaphase

Anaphase

Telophase

S Phase• How does DNA replicate?

Back to Interphase

THE CELL CYCLE

Prophase

MetaphaseAnaphase

Telophase

Cytokinesis

G1

S

G2

•Produces two identical copies of the chromosome during S phase of interphase•Catalyzed by many enzymes

DNA Replication

DNA Structure and composition: DNA strands are antiparallel, meaning they run

in opposite directions One strand runs in the 5' 3' direction, the other

in the 3' 5' direction

DNA Structure and composition:

The 3' end of one strand ends with the –OH on C3 (3rd carbon of the sugar molecule)

The 5' end of a strand ends with a phosphate group attached to the C5 (5th carbon of the pentose sugar)

DNA Replication Watson and Crick’s model of DNA structure

suggested how DNA could replicate Hydrogen bonds break Helix unzip Each strand act as a template to build new strand

At this time there were no experimental results to support this hypothesis

3 models of replication: Semiconservative replication would produce molecules

with both old and new DNA, but each molecule would be composed of one old strand and one new one.

Conservative replication would leave intact the original DNA molecule and generate a completely new molecule.

Dispersive replication would produce two DNA molecules with sections of both old and new DNA interspersed along each strand.

Semiconservative Model

DNA begins by unzipping itself the hydrogen bonds break between strands

2 strands separate, and each acts as a template or guide for directing the synthesis of the new strand.

"Semiconservative" is used to describe this process since each new DNA molecule contains 1 old strand and 1 new strand

This model was proven by Meselson & Stahl

Meselson and Stahl Experiment

Meselson and Stahl concluded that DNA replication is not conservative but semiconservative

Each strand acts as template for the building of the complementary strand

Each DNA strand is composed of one parent strand and one newly synthesized strand

Meselson-Stahl experiment E. coli bacteria was grown

using 15N, a denser isotope of N, for several generations.

When 15N DNA was centrifuged in cesium chloride, which forms a density gradient, all the DNA settled in one single layer, forming a band.

M and S then switched to 14N for one generation. If DNA replication was

conservative, they should have seen two bands, one containing the old DNA with 15N, the other containing the new DNA with 14N.

If DNA replication was semiconservative, they should have seen one intermediate band.

After two generations with 14N, they saw two bands: One intermediate band (mix of

15N and 14N) One lighter band (only 14N)

Over several generations, they found that the intermediate band did not go away even after they stopped using 15N. This indicates that the original 15N

DNA was still there, still acting as a template.

Meselson-Stahl experiment

Video Overview

http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/bio22.swf::Meselson and Stahl Experiment

DNA Replication

1. Separating the DNA strands2. Building Complementary

Strands 3. Linking of Nitrogenous Bases

and Proof-reading

Steps

1) Separating the DNA strands

The replication of DNA must take place in many steps. The entire DNA strand cannot be replicated at once because the unraveled DNA would be too large for the cell.

The piece of unwould DNA that is being replicated creates a DNA Replication Bubble

At each end of the bubble is the Replication Fork

The enzyme helicase breaks the hydrogen bonds holding the two complementary parent strands together, resulting in an unwound, unzipped helix that terminates at the replication fork.

The enzyme gyrase / topoisomerase relieves any tension from the unwinding of the double helix

1. Separating DNA Strands

Initiated at sites along DNA called Replication Origins – made of specific nucleotide sequences

Enzymes and other proteins work together to unwind and stabilize the double helix

•DNA helicase•DNA gyrase (aka topoisomerase)•SSBs (single-strand binding proteins)

The Players:

DNA Helicase

Recognizes specific nucleotide sequence (origin of replication)

Unwinds double helix by breaking H bonds between complementary base pairs

Opens up one or more replication bubbles

DNA Gyrase / Topoisomerase

Relieves the tension produced by the unwinding of DNA

Cuts one or two of the strands near the replication fork so that they can untangle and rejoin

Single-stranded Binding Proteins

SSBsKeep separated DNA strands

apart by blocking hydrogen bonding

Keep the templates (single DNA strands) straight

DNA Replication – Step 3

Single-stranded binding proteins (SSB’s) anneal to the newly exposed template strands, preventing them from reannealing by blocking hydrogen bonding.

1. DNA unwinding and unzipping

1. The enzyme helicase breaks the H-bonds holding the two complementary parent strands together, resulting in an unzipped helix that terminates at the replication fork.

2. The enzyme gyrase relieves any tension from the unwinding of the double helix.

3. Single-stranded binding proteins (SSBs) anneal to the newly exposed template strands, preventing them from re-annealing.

2) Elongation:Building of Complementary Strands

Replication begins in two directions from the origin

Toward direction of replication fork on one strand Away from direction of replication fork on the other

DNA polymerase builds new strands DNA polymerase III used in prokaryotes DNA polymerase III can only synthesize DNA

in the 5’ to 3’ direction

2. Building complementary strands

Because there may be more than one origin of replication in eukaryotes, more than one replication fork may exist

Link-replication animation: http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/bio23.swf

•Primase•DNA polymerase•DNA ligase

The Players

Primase

DNA nucleotides can only be added by DNA polymerase to an existing strand.

Primase builds RNA primers which are used to initiate DNA replication

DNA Replication DNA polymerase III cannot initiate a new strand by itself so a RNA

primer is required The enzyme primase lays down RNA primers that will be used by DNA

polymerase III as a starting point to build the new complementary strands.

DNA polymerase III

Takes free nucleotides found within the cell and adds them in the 5’ to 3’ direction, first to the RNA primer and then to the DNA nucleotide that was just added

The parent strand is used as a template

Leading (good guy) strand

The daughter strand that grows continuously towards the replication fork as the double helix unwinds

Occurs quicklyRequires only a single RNA

primer at replication origin

Leading Strand

DNA polymerase III adds the appropriate deoxyribonucleoside triphosphates to the 3 prime end of the new strand using the template strand as a guide.

The energy in the phosphate bonds is used to drive the process. The leading strand is built continuously toward the replication fork.

Lagging (scumbag) strand

The 3’ to 5’ parent strand is a problem for DNA polymerase since it must synthesize in the 5’ to 3’ direction!

Lagging strand

Built in short segments (in the 5’ to 3’ direction) away from the replication fork

Requires many RNA primers

Lagging Strand

A lagging strand composed of short segments of DNA, known as Okazaki fragments, is built discontinuously away from the replication fork.

DNA polymerase I

Removes the RNA primers once they have been used and replaces them with the appropriate DNA sequence

DNA ligaseJoins the Okazaki fragments into one

strand by the creation of phosphodiester bonds

Replacing the RNA Primers

DNA polymerase I excises the RNA primers and replaces them with the appropriate deoxyribonucleotides. DNA ligase joins the gaps in the Okazaki fragments by the creation of a phosphodiester bond.

Link-leading vs lagging animation: http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/micro04.swf

• DNA polymerase III acts as a proof-reader by checking the newly synthesized strand for any incorrectly inserted bases•If a mistake is found, it backs up and replaces the incorrect base with the correct one

3) Quality Control

DNA Replication – Final Step!

Errors missed by DNA polymerase III is “proofread” and repaired by DNA polymerase II by excising incorrectly paired nucleotides at the end of the complementary strand and adding the correct nucleotides.

Similar repair mechanisms also help to repair the damage caused by carcinogens (toxic chemicals, UV light and other radiation)

Termination The process of DNA replication ends when all the replication

bubbles meet. The DNA molecules rewind to gain their normal helical form. There is a problem though, due to the removal of primers- a little bit of a gap always remains on the lagging strand

Therefore, entire chromosome can never be fully copied!

It is estimated that with each round of duplication, around 100 bps of DNA are lost

To deal with this problem, each chromosome has a series of bases the essentially code for nothing tagged onto the end of the chromosomes. These buffer ‘ends’ are called Telomeres. Courtesy of the enzyme ‘telomerase’

Slow erosion of the telomeres (after approximately 50 replications) leads to the aging and death of the cell.

Animations

DNA Replication Fork: http://highered.mcgraw-hill.com/olcweb/cgi/pl

uginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/micro04.swf

How Nucleotides are Added: http://highered.mcgraw-hill.com/olcweb/cgi/

pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/bio23.swf

DNA Replication Simulator:

http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replication/index.html

Telomeres, Aging and Cancer

It is interesting to note that older people have shorter telomeres than younger people at the end of their chromosomes.

Certain types of cells have no apparent shortage to their telomeres ex. Sperm and cancer cells. Follow the links below to gather more information on telomeres:

http://learn.genetics.utah.edu/content/begin/traits/telomeres/

http://longevity.about.com/od/researchandmedicine/p/telomeres.htm