DNA Replication - Biochemistry - Lecture Slides, Slides of Biochemistry

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DNA Replication

• Basic vocabulary
• Initiation
• Replication forks
• DNA polymerases
• Strand differences
• Ligation
• Proof reading

Replication Vocabulary

• Semiconservative vs conservative
• DNA polymerase
• Theta structures
• Replication fork
• Unidirectional vs bidirectional
• Okazaki fragments
• Template strand
• Primer
• Leading strand
• Lagging strand
  • DNA ligase
  • RNA primers
  • Primase
  • Pol 1, Pol III
  • Processivity
  • Nick translation
  • Proofreading
  • Klenow fragment
  • Primosome
  • DNA binding proteins
  • 3’ and 5’ Exonucleases

Conservative Semiconservative

Heavy (^15) N

Light (^14) N (^14) N, 15 N

(^0) 1-3 (^) >10 (^0) 1-3 >

Meselson and Stahl CsCl

15 NH

4 Cl

Hybrid band Observed

Semiconservative

Cairns’ Replicating Fork

5’

5’ (^) 3’

3’ (^) 3’ 5’

Kornberg “DNA synthesis proceeds through the sequential addition of dNTP to the 3’ end of the growing chain of nucleotides

“The movement of the replicating fork is unidirectional, i.e., synthesis occurs on both strands but the fork is moving in only one direction

ERGO: Something is wrong Lagging Strand Okazaki fragments Leading Strand: Replication is in the direction of the moving fork

Theta structure

RNA Primer

SUMMARY OF DNA SYNTHESIS

dATP, dGTP, dCTP, dTTP + Template + Primer

A T C C G A T G A C T T

Template

U A G G

OH

3’ OH

C T A C T G A A

3’ end

DNA synthesis involves addition of dNTP

to 3’ end of a Primer as directed by a Template

Stages and Events in Replication

Initiation:

Proteins recognize the origin Parental strands separate Primosome initiates synthesis at replication fork

Elongation:

Replisome complex moves along DNA Leading strand is replicated continuously Lagging stand is replicated discontinuously

Parental strands unwind at the forks

Termination:

Duplicate chromosomes separate

Directionality

Loop

Replication must always proceed in a 5’ to 3’ direction

What is a Primosome? Ans: A 600 kD protein assembly composed of at least 7 individual proteins that take part in replication

Why is it needed?

What function does it perform?

Ans: Primosomes conduct the initial phases of replication, They unwind the DNA, separate and keep the strands apart, lay down an RNA primer

Ans: Because DNA polymerase cannot initiate the synthesis of a DNA chain, and leading and lagging strands must have a 3’OH to start off and extend

DNA Polymerases

Polymerase I

3’ - 5’ exonuclease

Polymerase II

Lagging strand fill in

Polymerase III

(low processivity)

(high processivity)

3’ - 5’ exonuclease

5’ - 3’ exonuclease to remove RNA primer

Leading and lagging strand DNA synthesis

DNA repair

Polymerase III

• Mwt = 130,
• 10 different polypeptide chains
• Core polymerase = , , and  subunits
• 2 molecules of  cause dimerization
•  and  allow pol III to bind  subunit
•  subunit forms sliding clamp that is

responsible for high processivity

• Assembled protein is called “holoenzyme”

Pol I only

Pol I, III

Absent in Klenow fragment

3’ 5’

5’ 3’

Proof Reading

New strand (clips from 3’ end)

Template strand

Proofreading

3’exonuclease

Bound dCTP

Template 3’ end