Enzymes and Proteins

1. Helicase

• Unwinds double helix
• Breaks hydrogen bonds between bases
• Creates replication fork

2. Single-Strand Binding Proteins (SSB)

• Bind to separated strands
• Prevent strands from reannealing
• Protect single-stranded DNA

3. Topoisomerase

• Relieves tension from unwinding
• Prevents supercoiling ahead of replication fork

4. Primase

• RNA polymerase enzyme
• Synthesizes short RNA primers (5-10 nucleotides)
• DNA polymerase needs primer to start

5. DNA Polymerase III (prokaryotes) / DNA Polymerase δ (eukaryotes)

• Main replication enzyme
• Adds nucleotides to 3' end only (5'→3' direction)
• Proofreads as it goes (3'→5' exonuclease activity)

6. DNA Polymerase I (prokaryotes)

• Removes RNA primers
• Replaces with DNA nucleotides
• 5'→3' exonuclease activity

7. DNA Ligase

• Seals gaps between Okazaki fragments
• Forms phosphodiester bonds
• Creates continuous strand

Leading vs. Lagging Strand

Leading Strand

• Synthesized continuously in 5'→3' direction
• Same direction as replication fork movement
• Only one primer needed

Lagging Strand

• Synthesized discontinuously in 5'→3' direction
• Opposite direction to fork movement
• Forms Okazaki fragments (1000-2000 nucleotides)
• Multiple primers needed
• Fragments joined by DNA ligase

Replication Steps

1. Initiation:

   • Helicase unwinds DNA at origin
   • SSB proteins stabilize
   • Primase adds RNA primers

2. Elongation:

   • DNA polymerase adds nucleotides (5'→3')
   • Leading strand: continuous synthesis
   • Lagging strand: Okazaki fragments formed

3. Termination:

   • DNA polymerase I removes RNA primers
   • Replaces with DNA
   • DNA ligase seals gaps
   • Two identical DNA molecules

Proofreading and Repair

Proofreading:

• DNA polymerase checks each nucleotide
• 3'→5' exonuclease removes errors
• Error rate: ~1 in 10 billion

Mismatch repair:

• Enzymes scan for mismatched bases
• Remove and replace incorrect nucleotides
• Occurs after replication

DNA repair mechanisms:

• Base excision repair
• Nucleotide excision repair (UV damage)
• Direct repair

Telomeres and Telomerase

Problem: DNA polymerase can't replicate ends of linear chromosomes

Telomeres:

• Repetitive sequences at chromosome ends (TTAGGG in humans)
• Protect genes from being lost
• Shorten with each division

Telomerase:

• Enzyme that extends telomeres
• Active in germ cells, stem cells
• Inactive in most somatic cells
• Overactive in cancer cells (immortality)

Key Concepts

1. Semiconservative: each new DNA has one old + one new strand
2. 5'→3' direction: DNA polymerase adds to 3' end only
3. Leading strand: continuous synthesis
4. Lagging strand: discontinuous, forms Okazaki fragments
5. Proofreading: ensures high fidelity (~1 error in 10¹⁰)
6. Telomeres: protect chromosome ends, shorten with age

Why "semiconservative":

• Each strand serves as template
• Base pairing (A-T, G-C) ensures accurate copying
• Original information conserved in each new molecule

$\boxed{\text{Parent DNA} \xrightarrow{\text{replication}} \text{2 hybrid DNAs (old + new strands)}}$

(b) Key Enzymes and Functions:

1. Helicase:

• Function: Unwinds double helix
• Breaks hydrogen bonds between base pairs
• Creates replication fork (Y-shaped structure)
• Uses ATP energy

2. Single-Strand Binding Proteins (SSB):

• Function: Bind to separated DNA strands
• Prevent strands from re-annealing
• Protect single-stranded DNA from nucleases
• Keep strands straight for replication

3. Topoisomerase (DNA Gyrase):

• Function: Relieves tension ahead of replication fork
• Cuts, untwists, and rejoins DNA
• Prevents supercoiling
• Without it: DNA would get too twisted and break

4. Primase:

• Function: Synthesizes RNA primers
• RNA polymerase (doesn't need primer itself)
• Makes short RNA sequences (~10 nucleotides)
• Provides 3'-OH for DNA polymerase to start

5. DNA Polymerase III (main enzyme in prokaryotes):

• Function: Synthesizes new DNA strand
• Adds nucleotides in 5' → 3' direction ONLY
• Requires 3'-OH group (needs primer)
• Has 3' → 5' exonuclease (proofreading)
• ~1000 nucleotides/second!

6. DNA Polymerase I:

• Function: Removes RNA primers
• Fills in gaps with DNA
• 5' → 3' exonuclease activity (removes primers)
• 5' → 3' polymerase activity (fills gaps)

7. DNA Ligase:

• Function: Seals nicks in sugar-phosphate backbone
• Joins Okazaki fragments
• Forms phosphodiester bonds
• Creates continuous strand

(c) Leading vs Lagging Strand:

Replication fork structure:

        5' ←——————— 3'  (parental)
       /              \
      /                \
    3' —————————————→ 5'  (parental)
   
   Leading strand →
   ← Lagging strand (Okazaki fragments)

Leading Strand:

• Synthesized continuously in 5' → 3' direction
• Same direction as replication fork movement
• Only ONE primer needed (at origin)
• DNA Pol III adds nucleotides smoothly
• No interruptions

Lagging Strand:

• Synthesized discontinuously in 5' → 3' direction
• Opposite direction to fork movement
• Requires MULTIPLE primers
• Made in short segments (Okazaki fragments)
  • Prokaryotes: 1000-2000 nucleotides
  • Eukaryotes: 100-200 nucleotides

Why the difference?

• DNA polymerase can ONLY synthesize 5' → 3'
• Two parental strands are antiparallel
• Fork moves in one direction
• Leading strand "lucky" - goes with fork
• Lagging strand "unlucky" - goes against fork, must be made backwards in chunks

(d) Processing Okazaki Fragments:

Step-by-step:

Step 1: Primase makes RNA primer

• Primase synthesizes short RNA primer (~10 nt)
• Provides 3'-OH for DNA Pol III

Step 2: DNA Pol III synthesizes Okazaki fragment

• Extends from primer in 5' → 3' direction
• Adds ~1000-2000 nucleotides (bacteria)
• Stops when it reaches previous primer

Step 3: DNA Pol I removes primer and fills gap

• 5' → 3' exonuclease removes RNA primer ahead
• Simultaneously fills gap with DNA
• "Nick translation" process

Step 4: DNA ligase seals nick

• Catalyzes phosphodiester bond formation
• Links 3'-OH of one fragment to 5'-phosphate of next
• ATP required (in eukaryotes) or NAD+ (in prokaryotes)
• Creates continuous strand

Detailed view:

Before processing:
5'—DNA—3' [RNA primer] 5'—DNA—3' [RNA primer] 5'—DNA—3'
                ↓
DNA Pol I removes primers:
5'—DNA—3'      5'—DNA—3'      5'—DNA—3'
        [gap]          [gap]
                ↓
DNA Pol I fills gaps:
5'—DNA—DNA—3'  5'—DNA—DNA—3'  5'—DNA—DNA—3'
           [nick]         [nick]
                ↓
DNA Ligase seals:
5'—DNA—DNA—DNA—DNA—DNA—DNA—DNA—DNA—3'
(continuous strand!)

Summary Table:

Proofreading and Error Rate:

3' → 5' exonuclease (proofreading):

• DNA Pol III checks each nucleotide added
• If mismatch: removes it, tries again
• Reduces errors from 1/10^5 to 1/10^7

Mismatch repair (post-replication):

• Separate system checks after replication
• Further reduces errors to 1/10^10
• Incredibly accurate!

$\boxed{\text{Replication: bidirectional, semiconservative, highly accurate (error rate } < 10^{-10})}$