Chapter 11: DNA Replication

During which phase of the cell cycle does replication occur?

• S phase (Synthesis phase) of interphase.

Prokaryotic replication

Column A – Terms

1. Leading strand

2. Okazaki fragments

3. Origin of replication (oriC)

4. Lagging strand

5. Replication forks
   

Column B – Definitions
A. Synthesized discontinuously.
B. Short DNA pieces on a lagging strand.
C. Starting point of DNA replication.
D. Sites where DNA is unwound.
E. Synthesized continuously.

• Origin of replication (oriC) – starting point.

• Replication forks – sites where DNA is unwound.

• Leading strand – synthesized continuously.

• Lagging strand – synthesized discontinuously.

• Okazaki fragments – short DNA pieces on a lagging strand.

Proteins/enzymes involved in prokaryotic replication:

Column A – Proteins/Enzymes

1. DNA helicase

2. DNA polymerase I

3. Topoisomerase/gyrase

4. DNA polymerase III

5. DNA ligase

6. DNA primase

7. SSB (single-strand binding proteins)
   

Column B – Definitions
A. Joins Okazaki fragments.
B. Lays down RNA primers.
C. Stabilizes single-stranded DNA.
D. Synthesizes most DNA.
E. Relieves supercoiling ahead of fork.
F. Unwinds the helix.
G. Replaces RNA primers with DNA.

• DNA helicase – unwinds the helix.

• SSB (single-strand binding proteins) – stabilize single-stranded DNA.

• DNA primase – lays down RNA primers.

• DNA polymerase III – synthesizes most DNA.

• DNA polymerase I – replaces RNA primers with DNA.

• DNA ligase – joins Okazaki fragments.

• Topoisomerase/gyrase – relieves supercoiling ahead of fork.

Replicative proteins (major ones and functions):

Column A – Proteins/Enzymes

1. Topoisomerase (gyrase in prokaryotes)

2. SSB proteins

3. DNA polymerase I

4. DNA ligase

5. Primase

6. DNA helicase

7. DNA polymerase III
   
   

Column B – Definitions
A. Synthesizes RNA primers.
B. Main enzyme for DNA elongation.
C. Prevents reannealing of single strands.
D. Seals nicks between Okazaki fragments.
E. Removes RNA primers, fills gaps with DNA.
F. Unwinds double-stranded DNA.
G. Relieves supercoils.

• DNA helicase: unwinds double-stranded DNA.

• SSB proteins: prevent reannealing of single strands.

• Primase: synthesizes RNA primers.

• DNA polymerase III: main enzyme for DNA elongation.

• DNA polymerase I: removes RNA primers, fills gaps with DNA.

• DNA ligase: seals nicks between Okazaki fragments.

• Topoisomerase (gyrase in prokaryotes): relieves supercoils.

Definitions 

Column A – Terms

1. Proofreading

2. Semi-conservative

3. Fidelity

4. Okazaki fragment

5. Bidirectional

6. Semi-discontinuous

7. Origin
   

Column B – Definitions
A. Accuracy of DNA replication.
B. Each daughter DNA molecule has one old strand and one new strand.
C. Replication proceeds in both directions from the origin.
D. Short DNA fragment synthesized on lagging strand.
E. Leading strand synthesized continuously, lagging strand synthesized in fragments.
F. Ability of DNA polymerase to correct errors during synthesis.
G. Specific sequence where DNA replication begins.

• Semi-conservative: Each daughter DNA molecule has one old strand and one new strand.

• Semi-discontinuous: Leading strand synthesized continuously, lagging strand synthesized in fragments.

• Bidirectional: Replication proceeds in both directions from the origin.

• Origin: Specific sequence where DNA replication begins.

• Okazaki fragment: Short DNA fragment synthesized on lagging strand.

• Fidelity: Accuracy of DNA replication.

• Proofreading: Ability of DNA polymerase to correct errors during synthesis.

What is the difference between DNA polymerase III and I in terms of their activities during DNA replication

• Pol III: synthesizes most of the new DNA (elongation).

• Pol I: removes RNA primers and fills in DNA (primer replacement).

What is the difference between an exonuclease versus an endonuclease? 

• Exonuclease: removes nucleotides from the ends of DNA strands.

• Endonuclease: cuts DNA internally at specific sequences.

What is processivity with respect to replication?

• The number of nucleotides added by a DNA polymerase per binding event before the enzyme dissociates from the template.

How is Okazaki processing done in prokaryotes versus eukaryotes? 

• Prokaryotes: DNA polymerase I removes RNA primers and fills in DNA; DNA ligase seals the fragments.

• Eukaryotes: RNase H removes RNA primers, DNA polymerase δ fills in DNA, DNA ligase I seals fragments.
  

Describe the end-problem of replication in eukaryotes. 

• Linear chromosomes cannot fully replicate their 5′ ends, leading to progressive shortening.

What are telomeres? Where are they located and what do they consist of? 

• Location: ends of linear chromosomes.

• Composition: repetitive DNA sequences that protect chromosome ends

Describe the activity of telomerase. What kind of enzyme is telomerase? 

• Function: elongates telomeres to prevent loss of genetic material.

• Type of enzyme: reverse transcriptase (RNA-dependent DNA polymerase).

What purpose does its snRNA serve? 

• snRNA purpose: serves as a template for adding repetitive DNA sequences to the chromosome ends.

What are the three major steps of PCR that allows for amplification of a DNA sequence of interest? 

1. Denaturation: separate DNA strands at high temperature.
   
   

2. Annealing: primers bind to target sequences.
   
   

3. Extension: DNA polymerase synthesizes new DNA from primers.

What goes into the PCR tube? 

• DNA template, primers, DNA polymerase (thermostable), dNTPs, buffer with Mg²⁺.

How is PCR similar to DNA replication in the cell? How is PCR different than DNA replication in the cell?

• Similarities: DNA polymerase extends a primer along a template; complementary base pairing dictates sequence.

• Differences: PCR is in vitro, uses thermal cycling, does not require helicase or SSB, and occurs on selected sequences rather than the entire genome.