DNA Replication and Mutations

DNA Replication Goals

Purpose of DNA replication
- Ensure genetic continuity across generations.
- Make an exact copy of DNA for cell division.
Process of DNA replication
- Involves multiple steps and enzymes to accurately replicate DNA.
Role of enzymes in DNA replication
- Facilitate and ensure accuracy in the replication process.

DNA Replication: The process by which a cell makes an exact copy of its DNA.
DNA Enzymes: Specific proteins that catalyze biochemical reactions in DNA processes.
Polymerase: An enzyme that synthesizes DNA molecules from nucleotides.

What is DNA replication?
- The process of copying DNA in a cell before it divides.
Where in the cell does DNA replication take place?
- Occurs in the nucleus of eukaryotic cells, and in the cytoplasm of prokaryotic cells.
What role do enzymes play in DNA replication?
- Enzymes like helicase, polymerase and ligase are crucial for unwinding DNA, synthesizing new strands, and sealing breaks.
What is the result of DNA replication?
- Two identical DNA molecules, each containing one original and one new strand (semi-conservative).

DNA Helicase: An enzyme that breaks hydrogen bonds between base pairs, separating the two DNA strands during replication.
Genetic Continuity: The process of maintaining genetic information across generations through DNA replication.
Semi-Conservative: Describes DNA duplication where the original double helix unwinds, and each strand serves as a template for a new complementary strand.
DNA Polymerase: Enzyme that joins free DNA nucleotides to form new strands by creating phosphodiester bonds, utilizing original strands as templates.
Free Nucleotides: DNA nucleotides available in the nucleus that pair with complementary bases on the template strand.
Joining: The process performed by DNA ligase to connect small DNA segments (Okazaki fragments) during replication on the lagging strand.

Formation:
- The bond between 5’ phosphate of one nucleotide and 3’ hydroxyl of another.
Structure:
- Alternating sugar-phosphate backbone.
- Base side groups attached to the backbone.
Properties:
- The hydrophilic nature of the backbone enables hydrogen bonding with the aqueous environment.

Definition:
- The mechanism by which a cell produces an exact copy of its DNA.
Timing:
- Occurs before cell division.
Importance:
- Ensures complete genetic instructions are passed to daughter cells for proper functioning.
- Reduces errors that might lead to cell malfunctions affecting growth and maintenance.

Complementary Base Pairing:
- Strands are antiparallel; 5’ to 3’ and 3’ to 5’ orientations.
- A-T pairs form two hydrogen bonds.
- G-C pairs form three hydrogen bonds.
- Phosphodiester bonds between nucleotides.
Characteristics:
- Semi-conservative replication, comprising one original (parent) strand and one newly synthesized strand.
Phases:
- Initiation, elongation, and termination.

Initiator proteins bind to origins of replication.
DNA helicase breaks hydrogen bonds between base pairs, unzipping the DNA into two strands.
Formation of a replication bubble with two replication forks moving bidirectionally.

Goal: Separate the two parent strands.
Helicase breaks hydrogen bonds, resulting in single-stranded template DNA on each side of the fork.

Single-strand binding proteins (SSBs) stabilize single-stranded templates.
Goal: Keep strands separated long enough for copying.
Function: Bind to exposed single-stranded DNA to prevent re-annealing or hairpin formation.

Primase lays RNA primers to provide a free 3’-OH group (as DNA polymerase cannot start de novo).
Goal: Establish starting point for DNA synthesis.

DNA Polymerase synthesizes new DNA in the 5’ to 3’ direction.
Golden rule: Reads template from 3’ to 5’ and synthesizes new DNA from 5’ to 3’.

Leading Strand:
- Template orientation: 3’ → 5’ toward the fork.
- Continuous synthesis with one RNA primer needed.
- DNA polymerase follows the fork smoothly.
Lagging Strand:
- Template orientation: 5’ → 3’ toward the fork.
- Discontinuous synthesis necessitating multiple RNA primers laid by primase.
- DNA polymerase extends each primer away from the fork in Okazaki fragments.

Removal of RNA primers and replacement with DNA.
Goal: Ensure final DNA contains no RNA segments.
Prokaryotes: DNA polymerase I acts as exonuclease to remove primers.
Eukaryotes: RNase H and FEN1 remove primer RNA, followed by gap filling by DNA polymerase.

DNA ligase seals the sugar-phosphate backbone.
Goal: Create a continuous strand by connecting fragments.
After primer replacement, “nicks” remain requiring formation of final phosphodiester bonds.

Goal: Maintain high accuracy during replication.
Many DNA polymerases possess 3’ to 5’ exonuclease activity for proofreading.
Mismatch repair systems fix any remaining errors following replication.

Goal: Finish replication properly and shut down machinery.
Prokaryotes (circular DNA): Forks meet at termination regions.
Eukaryotes (linear chromosomes): Forks meet as adjacent bubbles merge, with telomeres introducing complexity.

Definition: A change in the DNA sequence.
Point Mutations: Changes in a single nucleotide; typically occur during DNA replication and can be harmless or harmful.

Substitution: One nucleotide is replaced by another.
- May lead to synonymous (silent) changes or non-synonymous (functional change).
Insertion: An extra nucleotide is added.
Deletion: A nucleotide is omitted.
Frameshift: Insertions and deletions often shift the reading frame and can drastically affect protein function.

Impact: Point mutations can alter protein structure and function with effects ranging from mild to severe.
- Missense Mutations: An amino acid change due to substitution. Example: Sickle-cell anemia from a glutamic acid to valine substitution.
- Nonsense Mutations: Introduction of a premature stop codon resulting in truncated proteins. Example: Duchenne muscular dystrophy from a stop codon in the dystrophin gene.
- Frameshift Mutations: Result from inserts or deletes and can severely impair protein function due to shifts in reading frames. Usually more harmful than substitutions.