Biology Chapter 14 Part 2

Model of DNA Replication image

DNA Replications start at origins

Replication begins at specific DNA sequences called organs of replication

E coli (Prokaryote): One origin on its circular chromosome; two replication forks move bidriectionally

Eukaryotes: Many organs on each linear chromosome so large genomes can be copied effeinctlh

E Coli (Prokaryotes) DNA replication orgin

One organ on its circular chromosome; two replication forks move bilatterally

Eukaryotes DNA Replication Origin

Many origins on each linear chromosome so large can be copied efficiently

Opening and stabilizing the replication fork

At the origin, helicase separates the DNA strands by breaking hydrogen bonds

Single strand binding proteins keep the separated strands from reforming the helix

Topoisomerase relives twisting and supercoiling ahead of the replication fork.

Exam Q: Which enzyme separates (unzips) the two DNA strands during replication

Helicase

Single Binding proteins keep the separated from ______

reforming the helix

What does topoisomerase do?

Relieves twisting and supercoiling ahead of the replication fork

DNA polymerase Needs A Primer (slide)

• Primarse makes a short RNA primer that provides a free 3’- OH

• Dna polymerase III extends from the primer by adding complimentary DNA nucleotides

• DNA synthesis always proceeds 5→3’

• DNA polymerase I later removes RNA primers and replaces them with DNA

Don’t really need to know all of the primers

Want to unkown unzips, keeps them sticking, torsional sticking, adds new nucleotide

What does Primase do?

Comes in and makes a short RNA primer that provides a free 3’OH (important because otherwise DNA Polymerase won’t know where to start)

What does DNA Polymerase III do?

Extends from the primer by adding complementary DNA Nucleotides

What was does DNA synthesis always proceed?

5’ → 3’

What does DNA polymerase I do?

Later removes RNA primers and replaces them with DNA

Leading vs Laggins Strand Slide

• DNA polymerase can synthesize DNA only 5’ → 3’

• Because the template strands are antiparallel, the two new strands are made differently

• Leading strand: synthesized continuously toward the replication fork

• Lagging strand: synthesized discontinuously away rom the fork as Okazaki fragments.

DNA Polymerase can synthesize DNA only ’ - ‘

5’ → 3’

Because the template strands are antiparallel, the two new strands are made _____

differently

Leading Stand

synthesized continuously toward the replication fork

Lagging Strand

Synthesized discontinuously away from the Okazaki Fragments

How do leading and lagging strands differ?

Leading: continuous toward fork. Lagging: discontinuous away from fork.

Finishing the Lagging Strand

• Each Okazaki fragment begins with an RNA Primer

• DNA polymerase I removes each RNA primer and replaces it with DNA

• DNA ligase seals the remaining nicks, joining the fragment into one continuous strand

What are Okazaki fragments?

Short DNA sections synthesized on the lagging strand, each beginning with an RNA primer.

What do DNA polymerase I and DNA ligase do?

Polymerase I replaces RNA primers with DNA; ligase seals nicks between fragments.

Finishing the lagging strand: Each Okazaki fragment begins with an _____

RNA Primer

Finishing the lagging strand: DNA polymerase I removes each _______ _____ and replaces it with DNA

RNA primer

Finishing the lagging strand: DNA Ligase _____ the remaining nicks, joining the fragments into one continuing strand

Seals

Proofreading During DNA Replication

• DNA polymerase checks each newly added nucleotide as replication proceds

• If a base is mismatched, the incorrect nucleotide is removed and replaced

• Proofreading greatly lowers the DNA replication error rate

What is DNA proofreading?

DNA polymerase detects, removes, and replaces incorrect nucleotides during replication.

Proofreading During DNA Replication: DNA polymerase check each ____ _____ _____ as replication process

newly added nucleotide

Proofreading During DNA Replication: if a base pair is mismatched, what happens

the incorrect nucleotide is removed and replaced

_______ greatly lowers the DNA replication error rate

Proofreading

Mismatch Repair: Fixing Errors After Replication

• Some mismatches escape DNA polymerase proofreading

• Mismatch repair proteins identify the newly synthesized strand and remove the region containing the wrong base

• DNA polymerase fills the gape; DNA ligase seals it

What is mismatch repair?

After replication, proteins remove mismatched DNA; polymerase fills and ligase seals.

Nucleotide Excision Repair: Fixing DNA Damage

• Nucleotide excision repair removes damaged DNA rather than a simple replication mismatch

• UV light can create covalent links between adjacent pyridines, often called thymine dimers

• The damaged stretch is cut out, replaced by DNA polymerase, and sealed by DNA ligase

What is nucleotide excision repair?

Removes damaged DNA, such as UV-induced thymine dimers; polymerase and ligase repair it.

What does Nucleotide excision repair do?

Removes damaged DNA rather than a simple replication mismatch

Nucleotide Excision Repair: UV light can create covalent links between adjacent pyridines, often called ____ ______

thymine dimers

The End-Replicaiton Problem

• Eukaryotic chromosomes are linerar

• On the lagging strand, removal of the final RNA primer leaves no upstream 3’-OH for DNA polymerase

• Without a solution, chromosome ends become shorter after repeated rounds of replication

• Telomeres are repetitive, noncoding DNA that protect chromosome ends

What is the end-replication problem?

Removing the final lagging-strand primer leaves a gap, causing chromosome shortening.

The End-Replicaiton Problem: On the lagging strand, removal of the final RNA primer leaves _______

no upstream 3’-OH for DNA polymerase

The End-Replicaiton Problem: Without a solution, chromosome ends become _______

shorter after repeated rounds of replication

The End-Replicaiton Problem: ________ are repetitive, noncoding DNA that protect chromosome ends

Telomeres

What are telomeres?

Repetitive, noncoding DNA sequences that protect the ends of linear chromosomes.

Telomerase Extends Chromosome Ends slide

• Telomerase is a reverse transcriptase that carries its own RNA template

• It extends the 3’ end of the parental DNA strand with telomeric repeats

• Primase and DNA polymerase can then synthesize the complementary strand

• Telomerase is active in germ cells, many stem cells, and many cancer cells

What is telomerase

a reverse transcriptase that carries its own RNA template. It extends the 3’ end of the parental DNA strand with telomeric repeats. Primase and DNA polymerase can then synthesize the complementary strand. It is active in germ cells, many stem cells, and many cancer cells.

What does telomerase do, and where is it active?

Extends chromosome ends using its RNA template; active in germ, many stem and cancer cells.

• Telomerase is a __________ ____ transcriptase that carries its own ____ ________

Reverse transcriptase, RNA template

• _______ extends the 3’ end of the parental DNA strand with telomeric repeats

Telomerase

• Telomerase extends the 3’ end of the parental DNA strand with telomeric repeats

• Primase and DNA polymerase can then ______ the complementary strand

synthesize

• ______ is active in germ cells, many stem cells, and many cancer cells

Telomerase

PCR: Copying a specific DNA region in Vitro

• PCR = Polymearse chain reaction

• PCR amplifies the DNA between two known primer-binding sites

• Required: template DNA, two primers, dNTPs, and a heat stable DNA polymerase

What does PCR do?

• amplifies the DNA between two known primer-binding sites

What is required for PCR:

template DNA, two primers, dNTPs, and a heat stable DNA polymerase

PCR Uses Repeated Temperate Cycles

1. Denaturation: heat separates the DNA strands

2. Annealing: primers bind to complentary target sequeences

3. Extension: DNA polymerase synthesizes new DNA from each primer

• The cycle is related many times

First Step of PCR

Denaturation: heat separates the DNA strands

Second Step of PCR (after denaturation)

Annealing: primers bind (stick) to complentary target sequeences

Third Step (after annealing)

Extension: DNA polymerase synthesizes new DNA from each primer

PCR produces exponential amplification

• Ideally, the target DNA approximately doubles each cycle

• After n cycles, up to ~2ⁿ copies can be produced from one starting molecule

• Applications: diagnostics, forensics, genetic testing, cloning and research

How does PCR amplification increase each cycle?

Ideally doubles each cycle: 2ⁿ copies after n cycles from one starting molecule.

• Ideally, the target DNA approximately doubles each _____ cycle

PCR

PCR: After n cycles, up to ~____ⁿ copies can be produced from one starting molecule

2

Applications for PCR

diagnostics, forensics, genetic testing, cloning and research

Gel electrophoresis Separates DNA by size

• DNA is negatively charged and moves toward the positive electrode

• Agarose gel acts as a molecular sieve

• Smaller DNA fragments move through the gel faster and farther than larger fragments

• Compare bands with a DNA ladder to estimate fragment size

• A common use is checking the size of PCR products

How does gel electrophoresis separate DNA?

Negative DNA moves toward the positive electrode; smaller fragments travel farther. A ladder estimates size.

DNA Replication Image Overview

Helicase “The unzipping Enzyme”

This enzyme unzips the two strands of DNA, and breaks through the hydrogen bonds that hold the DNA bases together

DNA Polymerase “The Builder”

This enzyme replicates DNA molecules to build a new strand of DNA

Primase “The initializer”

DNA polymerase can’t figure out where to get started without a primer. This enzyme makes the primer (made of RNA) so that DNA polymerase can figure out where to go to start to work

Ligase “the gluer”

Seals the gaps between the Okazaki fragments to create one continuous DNA strand

Summary of DNA Replication Steps

1. DNA unwinds at the origin of replication.

2. Helicase opens up the DNA—-forming replication forks; these are extended bidirectionally.

3. Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the DNA.

4. Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling.

5. Primase synthesize (creates) RNA primers complementary to the DNA strand.

6. DNA polymerase starts adding nucleotides to the 3'-OH end of the primer.

7. Elongation of both the lagging and the leading strand continues.

8. RNA primers are removed by exonuclease activity.

9. Gaps are filled by DNA pol by adding dNTPs.

10. The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of phosphodiester bonds.