Today’s Outline

• Study Notes 6b Due!• Take up exam – View exam on Friday between 2-4pm in C-4.– Written appeals-process

• DNA Replication– Replication– PCR– Telomeres

• DNA Replication Tutorial 3-5pm Friday

Write Down:

What is one thing you learned about DNA replication from the readings?

To Understand DNA Replication, Understand the Parts

- Helicase- Topoisomerase- Single Strand Binding Proteins- DNA Polymerase III- Sliding Clamp- DNA Primase

HelicaseHelicase unzips DNA.

TopoisomeraseRelieves the tension produced when DNA is

unzipped.

Single Strand Binding Proteins

Stabilizes DNA and prevents it from binding to parallel strand.

DNA Polymerase III

Adds dNTPs to the newly synthesized strand on the leading strand. Adds in a 5’ 3’ direction.

Sliding Clamp

Holds DNA Polymerase III in place.

DNA Primase

Nucleotides can ONLY be added to the 3’ end of an already-attached nucleotide. A primer

provides this. DNA Primase builds the primer.

DNA

Polymerase Chain Reaction

• What does polymerase do?• What if….– I put an entire genome in a test tube.– I added DNA polymerase III and dNTPs.– I added very specific DNA primers that mapped

onto a specific gene.– Then I started heating and cooling the DNA over

and over and over and over….

Polymerase Chain Reaction5’

5’

3’

3’

5’

3’

5’3’

5’

3’

5’ 3’

- taq polymerase- dNTPs- primers- buffer

Discuss:

• Find three similarities and three differences between (i) DNA replication on the leading strand and (ii) DNA replication via PCR.

(Debrief using name cards)

The 1st Challenge of Lagging Strand Replication

• DNA Primase is used over and over• DNA Primase lays down ribonucleic acid.

Solution: an enzyme that can replace RNA nucleotides with DNA nucleotides DNA Pol1

The 2nd Challenge of Lagging Strand Replication

Solution: an enzyme that can connect 3’ carbons to phosphate groups

LIGASE

Ends of parental DNA strands

Leading strandLagging strand

Lagging strand

Last fragment Previous fragment

Parental strand

RNA primer

Removal of primers and replacement with DNA where a 3 end is available

Second round of replication

New leading strand

New lagging strand

Further rounds of replication

Shorter and shorter daughter molecules

5

3

3

3

3

3

5

5

5

5

53

DNA Pol1 Can only add nucleotides to the 3’ end of existing DNA.

Telomeres protect an organism’s genes from being eroded during successive rounds of DNA replication. In humans the sequence TTAGGG is repeated between 100 and 1000 times.

Which of these replaces RNA with DNA?

A) DNA Polymerase 1B) DNA Polymerase 3C) DNA PrimaseD) LigaseE) Nuclease

Which of these glues 3’ and 5’ DNA ends together?

A) LigaseB) DNA Polymerase 1C) Binding ProteinsD) DNA Polymerase 3E) DNA Primase

Which of these adds new nucleotides to the leading strand?

A) DNA Polymerase 3B) The Sliding ClampC) DNA Polymerase 1D) DNA PrimaseE) Binding Proteins

Which of these is not needed by the leading strand:

A) DNA Polymerase 1B) Binding ProteinsC) DNA PrimaseD) The Sliding ClampE) DNA Polymerase 3

Which of these is not needed by the lagging strand:

A) The Sliding ClampB) DNA Polymerase 1C) Binding ProteinsD) DNA Polymerase 3E) All are needed

Why are telomeres needed?

a) Primers can’t bind to 5’ ends of DNAb) DNA polymerase 1 can’t add nucleotides to 5’

ends of DNAc) Okazaki fragments don’t form at the end of

DNA strandsd) Helicase can’t bind to the end of DNA

strands.e) None of the above.

Write Down:

1. On a scale of 1 to 10, how well do you understand DNA replication?

2. What is one thing you understand really well?

3. What is one thing you do not understand?

Coming Up:1. Sanger Sequencing (Tues)2. Molecular Sculpting of DNA replication (Tues)3. Bonus assignment due (see Lecture Schedule)4. Field trip sign up5. DNA Replication Tutorial 3-5pm Friday