DNA Replication General Principles

Definition of DNA Replication
- The entire genome of a cell must be copied before the cell divides.
- Occurs during the S-phase of the cell cycle.
Key Characteristics of DNA Replication
- Speed:
- In bacteria: 5000 bp/sec
- In eukaryotes: 5000 bp/min
- Accuracy: Less than 1 error for every million bases.
Mutations and Alleles
- Changes in a gene sequence that affect product function are called mutations, which create new alleles.
Replicon
- A unit of DNA that is replicated, characterized by an origin of replication.
- Prokaryotic Cells: One circular chromosome with a single replicon.
- Eukaryotic Cells: Larger, linear chromosomes with multiple replicons.
- Example: Mouse (Mus musculus) has 20 pairs of chromosomes with approximately 25,000 replicons, averaging 150,000 bp in length.

Daughter Strand Synthesis

Semi-Conservative Nature of DNA Replication
- DNA replication begins with unwinding of the double helix at the origin of replication, forming a replication bubble with two forks.
- DNA (and RNA) is always synthesized in the 5' to 3' direction:
- The two strands in a DNA double helix are antiparallel, leading to opposite directions of daughter strand synthesis:
  - Continuous synthesis occurs on one strand towards the expanding fork, resulting in one long daughter strand.
  - Discontinuous synthesis occurs on the other strand, forming fragments oriented with their 3' ends away from the fork.

Prokaryotic DNA Replication

Origin of Replication:
- The bacterial single origin of replication (OriC) has sequences directing initiator protein binding.
- Cooperative binding of multiple DnaA initiator proteins to "DnaA boxes" triggers DNA strand separation at AT-rich DNA Unwinding Elements (DUEs).
- Strand separation permits access to single-stranded DNA by helicase and other proteins, forming a replication bubble.
Key Proteins Involved:
- DNA Helicase:
- Breaks hydrogen bonds between bases, moves along the lagging strand in a 5' to 3' direction toward the fork.
- Cannot open the double helix or initiate replication independently.
- Single-Strand Binding Proteins (SSBPs):
- Attach to each parental strand to prevent interactions between DNA strands and formation of secondary structures.
- Form tetramers (four proteins) that cover 35-65 nucleotides.
- DNA Gyrase:
- Type II topoisomerase that prevents torsional strain during DNA unwinding by cutting both strands of the double helix, allowing rotation before ligating them back together.
Daughter DNA Synthesis:
- Primase synthesizes short (10-12 nucleotide) complementary RNA primers using single-strand DNA as a template.
- DNA polymerases require a pre-existing polynucleotide with a 3'-hydroxyl to add nucleotides:
- DNA Polymerase III:
  - Adds DNA nucleotides to the 3'-end of the primer using the parental strand as a template.
  - Possesses 5' to 3' polymerase and 3' to 5' exonuclease activity for proofreading, correcting errors.
  - High processivity—synthesizes long polymers quickly.
- DNA Polymerase I:
  - Low processivity—removes RNA primers using 5' to 3' exonuclease activity while adding correct bases to an adjacent daughter strand using 5' to 3' polymerase activity.
- DNA Ligase:
  - Joins adjacent fragments by making a phosphodiester bond without adding a nucleotide.
- Terminator sequences (Ter) in some prokaryotes bind to terminator proteins (Tus) to halt replication fork expansion, ending replication.
Fidelity of DNA Replication:
- DNA polymerases have a high fidelity rate, with incorrect base incorporation at about 1 per 100,000 bp.
- Proofreading capability reduces errors to approximately 1 in 10 million bp.
- Mismatch repair proteins detect and repair errors after replication, significantly reducing mutation rates.

Eukaryotic DNA Replication

Key Differences from Prokaryotic Replication:
- Eukaryotic chromosomes are linear, presenting unique challenges.
- Larger eukaryotic genomes require multiple origins of replication, resulting in parallel replication bubbles.
- Eukaryotic DNA exists as part of chromatin, necessitating the assembly of newly synthesized DNA into nucleosomes with histones.
Eukaryotic Origins of Replication:
- Contain specific sequences that recruit origin recognition complex (ORC) proteins to initiate DNA replication.
- ORC initiator protein loads eukaryotic helicase onto the double helix at the origin during G1 phase; helicase separates strands during S-phase to open the replication bubble.
- Replication Licensing Factors bind origins, marking replication machinery action points during S-phase.
Eukaryotic Helicase:
- A complex of MCM2-7 proteins.
- Eukaryotes use type I Topoisomerase which cuts one strand to relieve torsional strain.
Daughter Strand Synthesis in Eukaryotes:
- DNA Polymerase Alpha:
- Has primase activity, synthesizing primer of 30-40 RNA nucleotides with DNA nucleotides added at the 3'-end.
- Lacks 3' to 5' exonuclease activity for proofreading.
- DNA Polymerase Delta:
- Part of a complex that synthesizes lagging strand DNA.
- DNA Polymerase Epsilon:
- Responsible for leading strand DNA synthesis.
- FEN1:
- A 5' to 3' exonuclease that removes RNA primers.
Histone Reassociation:
- Existing nucleosomes are disrupted by fork unwinding; histones reassociate with newly synthesized DNA, reforming nucleosomes on both double helices.

Telomeres

Problem of Linear Chromosome Replication:
- When a primer near the end of a linear strand is removed, there is no adjacent fragment to extend, causing chromosome shortening of about 70-100 bases with each round of replication.
Telomeres:
- Structures that protect chromosome ends from degradation, consisting of repeated sequences.
- With each cell division, telomeres shorten.
Telomerase:
- Contains a G-rich 3' overhang bound by telomerase enzyme, which includes a reverse complementary RNA sequence.
- Adds more repeats to lengthen the 3' overhang, allowing it to act as a template for synthesis of the second strand.
- Active in single-celled organisms and in germ cells of multicellular organisms.