DNA Replication and Repair

Overview and Biological Purpose of DNA Replication

• Before a cell splits in two, it copies all its DNA through DNA replication.

• This copying ensures each new daughter cell receives an exact, full set of genetic instructions.

• Replication happens during the cell cycle before chromosomes pack tightly and separate into the two new cells.

Structural Organization and Origins of Replication: Prokaryotes vs. Eukaryotes

• DNA copying begins at specific starting sequences called origins of replication (ORI).

• Starter proteins spot the origin sequence, stick to the DNA, and pull the two strands apart to open an unwound space called a replication bubble.

• Prokaryotes (such as Escherichia coli):

  • Contain a single circular chromosome.

  • Start copying at just one origin of replication.

  • Replication moves in both directions around the circle until two complete circular DNA copies are made.

• Eukaryotes (such as human cells):

  • Contain linear chromosomes stored in the nucleus.

  • Start copying at many origins of replication at the same time on each chromosome.

  • Replication bubbles expand in both directions and merge until two full linear DNA copies are made.

• Origin Sequence (ORI):

  • Top strand: $5'\text{-TACCATGGCCGATCTGAATTTAACCGACATG-}3'$

  • Bottom strand: $3'\text{-ATGGTAGCCGGCTAGACTTAAATTGGCTGTAC-}5'$

  • Initiator proteins recognize the middle bases of this sequence to pull the strands apart.

Models of DNA Replication

• The two strands of the original DNA separate, and each strand serves as a pattern (template) to build a matching new strand.

• Scientists considered three possible ways this could happen:

  • Semiconservative Model (the real mechanism):

  • Each parent strand separates and acts as a template for a new strand.

  • Every finished DNA double helix has exactly $1$ old parent strand and $1$ brand-new strand.

  • After round $1$, all molecules are half-old and half-new. After round $2$, half are hybrid and half are completely new.

  • Conservative Model:

  • The original parent DNA strands temporarily separate to direct synthesis, then rejoin completely.

  • One resulting double helix is completely original DNA, and the other is completely new DNA.

  • Dispersive Model:

  • The parent DNA is cut into pieces during copying and shuffled together with new pieces.

  • Every strand in every molecule ends up as a patchwork mix of old and new DNA.

Enzymatic Machinery and Replication Fork Components

• A replication fork is the Y-shaped zone at each edge of a replication bubble where DNA strands are actively separated.

• Key Bacterial Replication Proteins:

  • Helicase:

  • Sits at the point of the replication fork.

  • Unzips the double helix by breaking the hydrogen bonds holding base pairs together.

  • Single-Strand Binding (SSB) Proteins:

  • Coat the separated single strands immediately behind helicase.

  • Keep the exposed strands apart so they do not snap back together.

  • Topoisomerase:

  • Works on the DNA just ahead of the replication fork.

  • Relieves severe twisting strain by temporarily snipping, untwisting, and reconnecting the DNA backbone.

  • Primase:

  • Builds a short starter chain of RNA ($5\text{ to }10$ nucleotides long) called a primer.

  • Provides the exposed $3'\text{-OH}$ group that DNA-building enzymes must have to start.

  • DNA Polymerase III (DNA pol III):

  • The primary builder enzyme.

  • Reads the template strand and adds complementary DNA building blocks one by one to the $3'\text{-OH}$ end of a primer or existing strand.

  • DNA Polymerase I (DNA pol I):

  • The cleanup enzyme.

  • Cuts out RNA primer pieces and replaces them with matching DNA building blocks. It also assists in fixing errors.

  • DNA Ligase:

  • The sealing enzyme.

  • Joins broken ends of DNA strands together by creating permanent phosphodiester bonds along the backbone.

Directionality and Mechanism of Polymerization

• Direction Rules:

  • DNA polymerases always read template strands in the $3' \rightarrow 5'$ direction.

  • New DNA strands are always built in the $5' \rightarrow 3'$ direction.

• Starting Rules:

  • DNA polymerases cannot start from scratch; they can only add nucleotides to a pre-existing $3'\text{-OH}$ group.

  • Primase must first build an RNA primer on the template.

  • DNA Polymerase III then latches onto the primer's $3'\text{-OH}$ end to start making DNA.

Asymmetric Synthesis: Leading and Lagging Strands

• Because the two parent DNA strands are antiparallel (run in opposite directions), synthesis must happen differently on each side:

  • Leading Strand:

  • Built smoothly and continuously in the $5' \rightarrow 3'$ direction toward the unwinding fork.

  • Needs only $1$ RNA primer at the origin to get started.

  • Lagging Strand:

  • Built discontinuously in short pieces moving away from the fork.

  • These short segments are called Okazaki fragments.

  • Needs a new RNA primer laid down by primase for every single Okazaki fragment.

Okazaki Fragment Maturation, RNA Primer Removal, and DNA Ligation

• Okazaki fragments start out unlinked and contain RNA primers that must be replaced.

• Primer Replacement:

  • DNA Polymerase I acts as a nuclease to snip off the RNA primer from the $5'$ end of each fragment.

  • At the same time, it fills the empty spot with correct DNA nucleotides.

• Sealing the Gaps:

  • Replacing the primer leaves a tiny gap (nick) in the sugar-phosphate backbone.

  • DNA Ligase seals this nick by forming a chemical bond between the $3'\text{-OH}$ end of one fragment and the $5'\text{-phosphate}$ end of the next.

DNA Proofreading and Repair

• Cells keep copying errors extremely rare using built-in correction mechanisms:

  • Real-Time Proofreading:

  • DNA polymerases double-check each base pair ($\text{A-T}$ and $\text{G-C}$) the moment it is placed.

  • Mismatch Repair:

  • If an incorrect base is inserted, DNA Polymerase I cuts out the incorrect nucleotide and inserts the right one.

  • DNA Ligase then seals the repaired piece into the main DNA strand.

Step-by-Step Overview of Bacterial DNA Replication at the Fork

1. Helicase unwinds the double helix at the fork, separating the two strands.

2. SSB proteins bind to the single strands to keep them stable and separated.

3. DNA Polymerase III builds the leading strand continuously in the $5' \rightarrow 3'$ direction toward the fork.

4. Primase creates a short RNA primer for each Okazaki fragment on the lagging strand.

5. DNA Polymerase III extends the Okazaki fragment until it hits the previous primer, then moves to the next primer.

6. DNA Polymerase I removes the RNA primers and fills the open gaps with DNA nucleotides.

7. DNA Ligase joins the fragments by sealing the nicks in the sugar-phosphate backbone.