5.2 DNA Replication

SBI4U1

Humans rely on the continual regeneration of cells ( ie. Especially during injury)– Examples:• Humans begin as a single fertilized egg• Red blood cells, skin cells must regenerate continuously

The production of new cells is achieved throughMitosis & Cytokinesis

Recall: Cell Cycle

CELL CYCLE1) Growth stage:

Interphase2) Cell division:

production of 2 daughter cells

*note that DNA is copied during S phase of interphase in the cell cycle.

• In order for cells to divide they must:– Grow– Carry out metabolic activity– Replicate DNA

• During division, each daughter cell contains exact same genetic material as parent cell

DNA Replication: process of producing two identical DNA molecules from an original, parent DNA

There are 3 proposed models for DNA Replication:

1. Semi-conservative2.Conservative3.Dispersive

Models of DNA Replication1) Conservative model :

results in one new molecule and conserves the old

2) Semi-conservative model: results in two hybrid molecules of old and new strands.

3) Dispersive model : results in hybrid molecules with each strand being a mixture of old and new strands.

Now sit back and relax….

http://www.youtube.com/watch?v=TCuxp7tgb0g

Meselson and Stahl• Used different isotopes of N to label DNA in a cell– 14N (common) and 15N (rare, aka “heavy”)– Nitrogen was chosen b/c its an integral part of DNA– They were able to separate densities of isotopes

using centrifugation( more dense =sink to bottom, less dense= stay on top)

The Experiment:• They grew E. coli bacteria in a medium with 15N for 17

generations

• b/c N is in DNA, both strands of bacteria are labeled with 15N

• Some bacteria were transferred to a medium with 14N and reproduced

Observations:• DNA samples from 15N were uniform

• After 1st round of replication DNA was single band intermediate 15N and 14N

• After 2nd round of replication DNA was two band, half 15N and 14N and half 14N

• After more replication, two bands, similar to prior – supports SEMI CONSERVATIVE!!

Homework:

• Read and make notes 5.2• Complete Learning Checkpg. 222 #13-18

DNA REPLICATION

Initiation-Elongation-Termination

Recall:Semi-Conservative Replication

• Mechanism of DNA replication that produces two copies– both are made up of one new strand and one from original DNA ( parent strand)

• There are 3 basics phases in replication:1. Initiation2. Elongation3. Termination

Even though DNA replication is semi-conservative, due to complimentary base pairing, the two copies of DNA are identical.

1. Initiation• A portion of DNA double helix is unwound to

expose bases for new base pairing

• It begins at the origin of replication (specific nucleotide sequence).

• This is where the unwinding begins to form a “bubble” and as a result two replication forks are formed

As replication proceeds, each replication fork moves along the DNA in opposite directions.

Replication Fork

Initiation cont’d… Important Enzymes

• Helicase ( enzyme responsible for the unwinding of the double helix) It cleaves H- bonds b/t complimentary base pairs

• Other enzymes(SSBP) ensure new single strands do not “re-wind” into double helix

• Topoisomerase II helps to relieve strain on double helix section above replication fork

2. Elongation

Synthesis of 2 new DNA strands, each composed of 1 parent strand• DNA Polymerase III– Enzyme that adds new nucleotides to create

complementary strand of DNA– Begins on the free 3’ hydroxyl end of parent strand– Moves in 5’ to 3’ direction toward replication fork

(This is called the “leading strand”

Lagging Strand DNA polymerase III moves in opposite direction, away from replication

RNA Primer is short strand of RNA that is used to start or “prime” DNA replication– Primase is an enzyme that synthesizes RNA primer– Several primers are used to build lagging strand– Once each primer is added, new DNA fragments are

generated ( these are called OKAZAKI fragments)

• DNA Polymerase I removes RNA primer, fills gaps b/t Okazaki fragments

• DNA ligase joins the Okazaki fragments together

Okazaki Fragments

3.Termination

• Newly formed strand automatically rewind into double helix after replication

• The new DNA strands separate from each other

Animation: http://highered.mcgraw-hill.com/sites/dl/free/0072437316/120076/bio23.swf

Repairing Replication Errors

• Entire human DNA can be copied in a few hours-Errors are likely to occur, as so much is happening at

once• Examples of Errors:

1) Mispairing b/t 2 nucleotides ( ie. T paired wit G)2) Strand slippage causing additions/omissions of nucleotides

1) Mispairing Mispairing is when complimentary base pairs to not match up.

For example, G joins up with T.

This is due to flexibility in DNA structure and can accommodate misshaped pairings.

2) Strand Slippage

How do we fix errors in DNA Replication??

1) DNA Polymerase II enzymes “proofread” the newly synthesized DNA to catch any errors• Catches ~99% of mismatch 2)Mismatch repair– A group of enzymes recognize mispaired

nucleotides on the new strand– Replace it with correct nucleotide

…Beyond this, mutations can result

Comparing DNA Replication (pg. 227)

Prokaryotes Eukaryotes

***Use your textbook to complete the Venn diagram comparing DNA replication in Prokaryotes and Eukaryotes**

Learning Expectations...• Purpose of DNA replication• Different models for DNA replication• Meselson and Stahl’s Experiment & main findings• Semi-Conservative Replication

– Initiation– Elongation (leading vs. lagging strand/Okazaki fragments)– Termination

• Diagram of DNA replication• All enzymes involved in DNA replication• Errors in DNA replication• Mechanisms of repair for replication errors• Compare/contrast DNA replication in prokaryotes and

eukaryotes

Homework

• Pg. 229 # 3, 5, 6, 7, 8, 10, 11

• Quiz to follow on Chapter 5• Review h/w for Chapter 5: • pg. 235-236 pg. 238-239 # 1-14 ,16, 18-28 # 48, 52,72