Molecular genetics• Previous discussions focused on the individual.• Focus has now shifted to genes

• How are they encoded (3/29, 4/2)• How are they replicated (4/2, 4/5)• How are they expressed - transcription (4/7)• How are they expressed - translation (4/9) • Relationship between phenotype and genotype - pathways (4/12)

• How are they regulated (4/14, 4/16, 4/19)• How we study them - individual genes (4/19, 4/21, 4/23)• Review 4/28 and Exam 4/30

• How we study them - global studies (Genomics-4/26, 5/3, 5/5)• Final Exam 5/10

Important Dates in Determining the Structure of DNA

1 Why did they dothe experiment?

2How did they do the experiment?

3 What did theyfind out?

4 What did theylearn?

Model question

• One interpretation of the results obtained by Griffith after performing experiment (d, previous slide) is stated as the “conclusion” in previous slide.

State one other possible interpretation? What experiments and conclusion did they use to rule out this interpretation?

Review the findings that were used byWatson and Crick to propose the double helix model for the structure of DNA

–Erwin Chargaff’s data –Rosiland Franklin’s x-ray crystallography

data

Structure of DNA

• DNA is composed of four basic molecules; nucleotides– Nucleotides contain:• Phosphate• Sugar, deoxyribose

– Ribose in RNA

• One of four nitrogenous bases (two purines and two pyrimidines)

The two polynucleotide strands (the backbones) in the double helix run in opposite directions, and are said to be anti-parallel

5’-end

3’-end5’-end (free 5’-phosphate)

3’-end (free 3’-OH)

Because of the pairing (A-T; G-C), one polynucleotide chain is always complementary to the base sequence of the other strand

The strand of a DNA molecule has the base sequence 3’-GCCTTTAAG-5’. Upon replication the complementary base sequence on the other strand of DNA will be _____________.

Model questions

Which of the following is NOT a key feature of DNA molecule a. antiparallel b. minor groove

c. major groove d. double helix

*e. stems and loops

Matthew Meselson and Franklin Stahl, 1958

entirely new AND entirely old DNA moleculespresent

ALL DNA moleculesare made upof both oldand new DNA

entirely new DNA moleculespresent BUT not entirely old DNA molecules

What Meselson and Stahl expected if semiconservative replication

First generation results helped them rule out one of the three possible mode of replication

Matthew Meselson and Franklin Stahl, 1958

entirely new AND entirely old DNA moleculespresent

ALL DNA moleculesare made upof both oldand new DNA

entirely new DNA moleculespresent BUT not entirely old DNA molecules

Clincher evidence!Why?In dispersive model lighter DNA band should not have formed

Great test question:

Predict what the cesium chloride gradients would look like for conservative and dispersive replication!

123 4

1 = initiator proteins2 = single strand binding proteins3 = helicase4 = topoisomerase (gyrase)

The existence of leading and lagging strands during DNA replication is the result of the ___________________. a. need for an RNA primer to start DNA synthesis b. formation of Okazaki fragments

*c. fact that DNA polymerase can synthesize only in the 5’ to 3’ direction d. semi conservative nature of DNA replication e. None of these.

Model questions

Replication• DNA Polymerase III

– Synthesize new DNA in the 5’ 3’ direction• Synthesizes long sequences of new DNA• Is highly processive; synthesizes DNA for a long period of time

without releasing the template• For example, synthesizes leading strand

• DNA Polymerase I– Synthesize new DNA in the 5’ 3’ direction

• Only synthesizes short sequences of new DNA• But before it could do this, it needs to remove RNA primers• This is achieved by its 5’ 3’ exonuclease activity

Replication

• DNA Polymerase I– Synthesize new DNA in the 5’ 3’ direction

• Only synthesizes short sequences of new DNA

– 3’ 5’ exonuclease activity (proofreading)– 5’ 3’ exonuclease activity (remove primers)

• DNA Polymerase III– Synthesize new DNA in the 5’ 3’ direction

• Synthesizes long sequences of new DNA

– 3’ 5’ exonuclease activity (proofreading)

NOTE: DNA polymerase III does not have the 5’ 3’ exonuclease activity

5’ 3’ exonuclease activity of DNA polymerase I

Model question

You know that DNA ligase seals the gaps left behind by DNA polymerase I and completes DNA replication. You alsoknow that both enzymes form the phosphodiester bonds. If that is the case why do cells need DNA ligase to seal the nicks and why not they use DNA polymerase I to seal these nicks?