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Chapter 12: DNA Replication and Recombination

Lectures 19 and 20

Problem set 3A: due at beginning of lecture on Monday, Oct. 16th

DNA Replication is semiconservative

Meselson-Stahl experiment: 15N-labeling and CsCl density gradient centrifugation.

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Overview of replication

Components required in vitro:1. Enzyme: DNA polymerase2. Template: DNA3. deoxyribonucleotide triphosphates

(dNTP's = dATP, dCTP, dGTP, and dTTP)4. Primer

(Animation: Nucleotide Polymerization by DNA Polymerase.)(To view animations go to the web site for the text book:http://bcs.whfreeman.com/pierce2e/default.asp?s=&n=&i=&v=&o=&ns=0&uid=0&rau=0 This is the same site that is listed in the Course Logistics document from the beginning of the course.Click on: “Genetics in Motion: Interactive Animations”)

General Features of Replication

1. Synthesis is always in the 5'-->3' direction.2. Starts at a specific point: "origin of replication" (=ori)3. Is usually bidirectional (sometimes unidirectional)4. Bacterial chromosome and plasmids each have one ori5. Eukaryotic chrom's have many ori's. because they’re so long!

(Animation: Bidirectional Replication of DNA)

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Rolling circle replication, a type of replication done by phiX174 and some other viruses

Linear chromosomes have multiple origins of replication.

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Some enzymes to get to know--the big guys in bacterial replication.

Bacterial Replication Enzymes to Know:

A. Binds to origin and separates strands of DNA to initiate replication.

B. DNA gyrases (topoisomerases): reduce supercoiling of the DNA ahead of replication fork by making, then resealing breaks in the DNA backbone.

C. Helicases: Denature DNA at replication fork.

D. Single-strand binding proteins : keep DNA single stranded.

E. Primase: an RNA polymerase that uses DNA as a template to make RNA primer for DNA replication.

F. DNA polymerase III (at least 10 different polypeptide subunits):elongates primers in the 5'--->3' direction, giving continuous synthesis on the leading strand and discontinuous synthesis on thelagging strand.

G. DNA polymerase I: digests RNA primers with its 5'--->3' exonuclease activity and replaces with DNA.

H. DNA ligase: joins unlinked DNA strands by sealing nicks in the sugar-phosphate backbone.

I. DNA polymerase II: involved in DNA repair.

DNA Replication proteins in eukaryotic cells have similar functions

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Deoxyribonucleoside triphosphates are incorporated into the growing DNA chain.

Animation: Coordination of Leading and Lagging-Strand Synthesis

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Animation: Overview of Replication

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During replication DNA forms a loop to allow two DNA polymerase III complexes to work together.

The fidelity of DNA replication is controlled at more than three levels.Error rate: <1 nucleotide/billion!

1. Accurate nucleotide selection 2. DNA proofreading

A. Incorrect bases don’t fit well.

B. Many DNA polymerases pause if incorrect base is inserted.

C. If they have 3’ 5’ exonuclease activity, they can cut off the incorrect base

D. The correct base can now be inserted.

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3. Mismatch repair: recognition of mismatched bases after replication is complete.

Selective repair of the new strand, becauseThe old strand is recognized because some nucleotides on it are methylated.

(more on repair of DNA damage latter in course)

Telomeres and the end replication problem.Telomerase: a ribonucleoprotein (RNA and protein) reverse transcriptase

1. Its RNA has sequence complementary to telomere sequence.

2. Uses its own RNA as template.

3. Adds additional telomeric repeatsto the ends of telomeres.

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1. Shortening of telomeres appears to contribute to cell death and aging.A. germ-line cells: high telomerase activityB. somatic cells: very low telomerase activity

2. Telomerase negative mice: premature aging after a # of generations.

3. Somatic cells engineered to have telomerase activity: divide indefinitely.

4. Telomerase activation is part of the development of most advanced cancers.

Recombination

I Involves proteins that:1. nick DNA2. help complementary strands base pair in new combos3. resolve intermediate structures formed by further nicking and

ligation.4. probably do small amounts of strand synthesis

II The Holliday model.

III The Double-strand-break repair model

(Animation: Mechanism of Homologous Recombination)

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The Holliday Model

1. Single strand nicking

2. Single strand invasion leads toformation of heteroduplex DNA

3. Migration of Hollidayjunction

4. Rotation of molecules

5. Removal of Holliday junctions by DNAse cutting

6. The type of cutting affects whether recombinants are seen or not

DS break repair model(more likely)

Differences:1. Both strands of 1 molecule break.

2. Strand digestionand strand extensionoccur.

3. Junctions do not slide.

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How does all this information get out of the DNA into the cell?