Lecture 5 DNA replication Slides

In vivo DNA replication

DNA replication that happens inside a living cell.

In vitro DNA replication

DNA replication that happens outside of a cell, such as PCR in a lab tube.

Why should in vivo and in vitro replication be separated when studying?

In vivo replication uses the full cellular replication machinery, while in vitro PCR uses a simpler lab setup with DNA, primers, polymerase, dNTPs, and heat.

DNA polymerase III

The main replicative DNA polymerase in E. coli.

Pol III holoenzyme

The full E. coli replication complex that copies both the leading and lagging strands at the replication fork.

Pol III core

The part of DNA polymerase III that contains the polymerase and proofreading activities.

Alpha subunit of Pol III

The subunit responsible for DNA polymerase activity.

Epsilon subunit of Pol III

The subunit responsible for 3' to 5' proofreading exonuclease activity.

3' to 5' exonuclease activity

Proofreading activity that removes incorrectly added nucleotides from the growing DNA strand.

Beta sliding clamp

A ring-shaped protein that keeps DNA polymerase attached to DNA.

Function of the beta sliding clamp

It increases processivity by preventing DNA polymerase from falling off the DNA template.

Clamp loader

The protein complex that loads the beta sliding clamp onto DNA.

Processivity

The ability of DNA polymerase to add many nucleotides without detaching from the DNA.

Replisome

The group of proteins that work together at the replication fork to copy DNA.

Replication fork

The Y-shaped region where parental DNA strands separate and new DNA strands are synthesized.

Leading strand

The new DNA strand synthesized continuously in the same direction as replication fork movement.

Lagging strand

The new DNA strand synthesized discontinuously in short fragments opposite the direction of fork movement.

Why is one strand continuous and the other discontinuous?

DNA polymerase can only synthesize DNA 5' to 3', but the two template strands are antiparallel.

Okazaki fragments

Short DNA fragments synthesized on the lagging strand.

Why are Okazaki fragments formed?

Because the lagging strand must be copied in short pieces while still following the 5' to 3' synthesis rule.

Helicase

The enzyme that separates the two parental DNA strands at the replication fork.

DnaB

The E. coli helicase that unwinds DNA during replication.

What direction does DnaB move?

DnaB moves 5' to 3' along the single-stranded DNA it is bound to.

Why does helicase need ATP?

ATP provides the energy for helicase to move along DNA and separate the strands.

Topoisomerase

An enzyme that relieves twisting stress and supercoiling ahead of the replication fork.

DNA gyrase

The main E. coli topoisomerase used during DNA replication.

Why is topoisomerase needed during DNA replication?

Helicase unwinding causes the DNA ahead of the fork to become overwound, so topoisomerase relieves that stress.

Primase

An RNA polymerase that makes short RNA primers for DNA replication.

DnaG

The E. coli primase that makes RNA primers during DNA replication.

Why is primase needed?

DNA polymerase cannot start DNA synthesis from nothing, so primase makes a primer that DNA polymerase can extend.

RNA primer

A short RNA strand that provides a free 3' OH for DNA polymerase.

Why does DNA polymerase need a 3' OH?

DNA polymerase adds new nucleotides onto the 3' OH of a preexisting strand.

How many primers are usually needed for the leading strand?

Usually one primer is needed to start leading-strand synthesis.

Why are many primers needed for the lagging strand?

Each Okazaki fragment needs its own RNA primer.

DNA polymerase I

The E. coli enzyme that removes RNA primers and replaces them with DNA.

RNase H

An enzyme that removes RNA that is base-paired with DNA.

Why can RNase H not finish primer removal alone?

It cannot remove the final RNA nucleotide attached to DNA, so another enzyme such as Pol I is needed.

DNA ligase

The enzyme that seals nicks in the DNA sugar-phosphate backbone.

What does DNA ligase do after primer replacement?

It joins DNA fragments together by sealing the remaining nick.

Why does ligase act after primer replacement?

Ligase seals DNA-to-DNA nicks, so the RNA primer must be removed first.

SSB protein

Single-stranded DNA-binding protein.

Function of SSB

SSB binds single-stranded DNA, protects it, and prevents it from forming secondary structures.

Why is SSB important at the replication fork?

It stabilizes the separated DNA strands so they can be copied.

Main proteins needed at the replication fork

Helicase, topoisomerase, primase, DNA polymerase III, beta sliding clamp, clamp loader, SSB, Pol I, RNase H, and ligase.

Basic sequence of replication fork activity

Helicase unwinds DNA, topoisomerase relieves supercoiling, SSB protects single strands, primase makes primers, Pol III extends DNA, Pol I/RNase H remove RNA primers, and ligase seals nicks.

What happens first at the replication fork?

Helicase separates the parental DNA strands.

What happens after helicase unwinds DNA?

Topoisomerase relieves supercoiling and SSB protects the single-stranded DNA.

What enzyme makes the primer during replication?

Primase.

What enzyme extends the primer during E. coli replication?

DNA polymerase III.

What enzyme replaces RNA primers with DNA?

DNA polymerase I.

What enzyme seals the DNA backbone?

DNA ligase.

Why is the lagging strand more complicated than the leading strand?

It must be made in many short fragments that each require priming, extension, primer removal, and ligation.

Trombone model

The model explaining how lagging-strand DNA forms loops during replication.

Why does the lagging strand form loops in the trombone model?

It lets the lagging-strand polymerase stay with the replisome while synthesizing DNA opposite the direction of fork movement.

What happens to the lagging-strand loop as an Okazaki fragment is synthesized?

The loop grows as the Okazaki fragment gets longer.

What happens when an Okazaki fragment is complete?

The polymerase releases the completed fragment and moves to a new primer.

Why can both DNA strands be replicated at the same time?

The Pol III holoenzyme contains multiple polymerase cores that can work on the leading and lagging strands together.

How does the replisome solve the antiparallel strand problem?

It uses continuous synthesis on the leading strand and looped discontinuous synthesis on the lagging strand.

What does "DNA synthesis is 5' to 3'" mean?

New nucleotides are added to the 3' end of the growing DNA strand.

What is the template direction for DNA polymerase?

DNA polymerase reads the template strand 3' to 5' while synthesizing the new strand 5' to 3'.

What did the DnaB helicase experiment determine?

It determined the direction that DnaB moves along single-stranded DNA.

How did scientists determine DnaB helicase direction?

They used labeled DNA fragments on opposite ends and observed which fragment was displaced.

What did the DnaB experiment show?

DnaB translocates 5' to 3' along single-stranded DNA.

Why was ATP included in the helicase experiment?

Helicase needs ATP to move and unwind DNA.

What happened when ATP was absent in the helicase experiment?

No unwinding occurred.

PCR

Polymerase chain reaction, a lab technique used to amplify a specific DNA sequence.

What type of DNA replication is PCR?

In vitro DNA replication.

Main purpose of PCR

To make many copies of a specific DNA segment.

What does PCR amplify?

The DNA sequence located between two primers.

Why does PCR only amplify a specific region?

The primers bind to specific sequences flanking the target DNA.

PCR template DNA

The DNA sample that contains the target sequence to be amplified.

PCR primers

Short synthetic DNA oligonucleotides that bind to opposite strands around the target region.

dNTPs

The free DNA building blocks used to synthesize new DNA.

DNA polymerase in PCR

The enzyme that extends primers to copy the target DNA.

Heat-stable polymerase

A polymerase that remains functional after high-temperature DNA denaturation.

Taq polymerase

A heat-stable DNA polymerase from Thermus aquaticus.

Thermus aquaticus

A thermophilic bacterium that lives in hot environments and produces Taq polymerase.

Why is Taq polymerase important for PCR?

It survives the repeated heating steps used to denature DNA.

What was the problem with early PCR polymerases?

They were destroyed by heat and had to be replaced after each cycle.

How did Taq polymerase improve PCR?

It allowed PCR to be automated because the polymerase did not need to be replaced each cycle.

Kary Mullis

The scientist credited with inventing PCR.

What disease mutation was PCR first reported to help detect?

The beta-globin mutation that causes sickle-cell anemia.

PCR denaturation

The heating step that separates double-stranded DNA into single strands.

PCR annealing

The cooling step where primers bind to complementary DNA sequences.

PCR extension

The step where DNA polymerase extends the primers and copies the target DNA.

Three steps of PCR

Denaturation, annealing, and extension.

Why does PCR use heat instead of helicase?

Heat separates the DNA strands in the test tube, so helicase is not required.

Why does PCR use primers?

DNA polymerase cannot start synthesis de novo and needs a preexisting 3' end.

How should PCR primers face?

Their 3' ends should point toward each other.

Why must PCR primers point toward each other?

So DNA polymerase copies the DNA region between them.

Why are primers more likely to bind than the original DNA strands reanneal?

Primers are present at a much higher concentration than the template DNA strands.

Why is PCR called a chain reaction?

The DNA products of one cycle become templates in the next cycle.

Why is PCR exponential?

Each cycle approximately doubles the amount of target DNA.

How many cycles are commonly used in PCR?

About 25 to 30 cycles.

How much amplification can happen after 20 ideal PCR cycles?

About 2^20, or roughly one million-fold.

What remains mostly unamplified during PCR?

DNA outside the region defined by the primers.

Why is PCR highly sensitive?

It can amplify very tiny amounts of DNA.

What is a risk of PCR sensitivity?

Contaminating DNA can also be amplified.

What are common PCR uses?

Cloning, DNA analysis, forensics, disease diagnosis, ancient DNA studies, and detecting infections.

Reverse transcriptase PCR

A PCR-related method where RNA is first converted into DNA.