DNA Replication: Models and Accuracy

DNA Replication Accuracy

• Replication must be extremely accurate to avoid catastrophic consequences.
• One error per million base pairs can lead to 6400 mistakes each cell division.
• Replication is also very fast
• E. coli replicates DNA at a rate of 1000 nucleotides per second.

DNA Replication Models

• There are 3 proposed models of DNA replication: conservative, dispersive, and semiconservative.
• In the conservative model, the entire original DNA molecule serves as a template for a completely new molecule.
• In the dispersive model, regions of both strands of the original DNA molecule are interspersed with regions of new DNA in both strands of the new molecule.
• In the semiconservative model, the 2 original DNA strands separate, and each strand serves as a template for a new strand.

Meselson and Stahl Experiment

• Meselson and Stahl tested the 3 models of DNA replication in E. coli.
• They used 2 nitrogen isotopes: the common form, 14N, and a rare, heavy form, 15N.
• First, E. coli were grown in 15N media and then transferred to 14N media.
• Cultured E. coli were then subjected to equilibrium density gradient centrifugation
• After one round of replication, the DNA appeared as a single band at intermediate weight.
• After a second round of replication, the DNA appeared as 2 bands, one light and one intermediate in weight.
• These results support the semiconservative replication model.

Modes of Replication

• Replicons are units of replication that contain a replication origin
• Theta replication occurs in circular DNA with a single origin and forms a replication fork, typically proceeding bidirectionally.
• Rolling-circle replication can be found in viruses and the F factor of E. coli and features a single origin of replication.
• Linear eukaryotic replication happens in in eukaryotic cells, with thousands of origins and a replicon size of ~200,000–300,000 bp.

Linear Eukaryotic Replication Requirements

• Linear eukaryotic replication necessitates a template strand, raw nucleotide materials, and certain enzymes and other proteins.
• DNA polymerase adds nucleotides exclusively to the 3' end of the extending strand, meaning replication proceeds only in the 5' to 3' direction.
• Replication is continuous on the leading strand and discontinuous on the lagging strand.
• Okazaki fragments consist of discontinuously synthesized short DNA sequences forming the lagging strand.

Characteristics of Theta, Rolling-Circle, and Linear Eukaryotic Replication

• Theta replication uses a circular template without nucleotide strand breakage, has 1 replicon, and may be unidirectional or bidirectional, resulting in 2 circular molecules.
• Rolling-circle replication employs a circular template, involves nucleotide strand breakage, has 1 replicon, is unidirectional, and yields one circular and one linear molecule.
• Linear eukaryotic replication utilizes a linear template, does not involve nucleotide strand breakage, has many replicons, is bidirectional, and results in 2 linear molecules.

Bacterial Replication: Initiation and Unwinding

• Bacterial DNA replication initiates at a 245 bp sequence in the oriC (single origin replicon).
• This process requires an initiator protein, DnaA in *E. coli
• Unwinding also needs an initiator protein and DNA helicase.
• Single-strand-binding proteins (SSBs) and DNA gyrase (topoisomerase) are also required.

Bacterial Replication: Elongation

• Elongation is carried out by DNA polymerase III.
• Primers consist of existing RNA nucleotides with a 3'-OH group for new nucleotide additions and are typically 10–12 nucleotides long
• Primase is an RNA polymerase.
• The leading strand requires a primer only at the 5' end.
• The lagging strand requires a new primer at the beginning of each Okazaki fragment.

Bacterial Replication: Elongation Requirements

• An active replication fork necessitates:
  • DNA helicase to unwind DNA.
  • Single-strand-binding proteins to protect single nucleotide strands.
  • DNA gyrase to relieve strain.
  • Primase to synthesize RNA primers w/ a 3'-OH group.
  • DNA polymerase to produce leading and lagging nucleotide strands.

Bacterial Replication: Termination

• Elongation is carried out by DNA polymerase III.
• RNA primers are removed via DNA polymerase I and connecting the nicks after RNA primers are removed, via DNA ligase.
• Termination occurs when replication forks meet or by a termination protein.

Characteristics of E. coli DNA Polymerases

• DNA polymerase I removes and replaces primers.
• DNA polymerase II is involved in DNA repair and restarts replication, but halts synthesis.
• DNA polymerase III elongates DNA.
• DNA polymerase IV and V participate in DNA repair; translesion DNA synthesis.
• All DNA polymerases synthesize any sequence specified by the template strand with the help of dNTPs to synthesize new DNA
• They synthesize in the 5' - 3' direction by adding nucleotides to a 3'-OH group, and require a 3'-OH group to initiate synthesis and need other proteins
• They catalyze the formation of a phosphodiester bond by joining the 5 -phosphate group of the incoming nucleotide to the 3 -OH group of the preceding nucleotide on the growing strand.
• They produce newly synthesized strands that are complementary and antiparallel to the template strands
• All polymerases has 5’ - 3’ polymerase activity but differ in exonuclease activity
• DNA polymerase I has both 3’ - 5’ and 5’ - 3’ exonuclease activity
• DNA polymerase II and III have 3’ - 5’ exonuclease activity
• DNA polymerase IV and V have no exonuclease activity

Main Components of Bacterial Replication

• Initiator proteins binds the origin to separate DNA strands.
• DNA helicase unwinds DNA at the replication fork.
• Single-strand-binding proteins attach to single-stranded DNA and prevent secondary structures.
• DNA gyrase moves ahead of the replication fork and reseals breaks to release torque.
• DNA primase synthesizes a short RNA primer to provide a 3'-OH group for DNA nucleotides.
• DNA polymerase III elongates a new nucleotide strand from the 3' end.
• DNA polymerase I removes RNA primers and replaces them with DNA.
• DNA ligase joins Okazaki fragments by sealing breaks in the sugar-phosphate backbone.

The Fidelity of DNA Replication

• DNA polymerase I's 3' to 5' exonuclease activity removes incorrectly paired nucleotides (proofreading).
• Mismatch repair corrects errors after replication.

Eukaryotic DNA Replication Initiation

• Eukaryotic DNA replication begins with autonomously replicating sequences (ARSs) that measure 100–120 bps.
• The origin-recognition complex (ORC) binds to ARSs to start DNA replication.
• ORC recruits and loads helicase, and eukaryotes don't require an initiator protein.

Licensing of Eukaryotic Replication

• Licensing ensures DNA replication by the licensing factor.
• ORC attaches to each origin of replication and joins with licensing factors to form MCM (minichromosome maintenance).
• It involves eukaryotic DNA polymerase.

Eukaryotic DNA Polymerases

• DNA polymerase alpha initiates nuclear DNA synthesis and DNA repair and has primase activity.
• DNA polymerase delta participates in lagging-strand synthesis of nuclear DNA, DNA repair, and translesion DNA synthesis.
• DNA polymerase epsilon leads strand synthesis.
• DNA polymerase gamma replicates and repairs mitochondrial DNA.
• DNA polymerase zeta, eta, iota, and kappa participate in translesion DNA synthesis.
• DNA polymerase theta and lambda participates in DNA repair.
• DNA polymerase mu and sigma possibly participates in nuclear DNA replication, DNA repair, and sister-chromatid cohesion
• DNA polymerase phi (φ) and Rev1 participates in translesion DNA synthesis.

Creation of Nucleosomes in Eukaryotic Replication

• Creation of nucleosomes requires original nucleosome disruption on the parental DNA, redistribution of preexisting histones, and new histones being synthesized.

The End-Replication Problem and Telomeres

• Replication around the circle provides a 3'-OH group in front of the primer, nucleotides can be added when the primer is replaced.
• Linear DNA telomeres, the terminal primer, are positioned 70-100 nucleotides from the chromosome's end, leaving a gap that can't be replicated.
• To solve the end-replication problem, telomerase extends DNA, filling gaps from RNA primer removal.
• Telomerase contains a protruding end w/ a G-rich repeated sequence and binds it, with the help of an RNA template.
• Nucleotides are added to the 3' end of the G-rich strand and after several nucleotides have been added, the RNA template moves along the DNA,
• More nucleotides are added and then the telomerase is removed.
• Synthesis takes place on the complementary strand, filling in the gap due to the removal of the RNA primer at the end.

Recombination

• Homologous recombination involves the exchange of homologous DNA molecules during crossing over for genetic variation and DNA repair
• Recombination utilizes the Holliday junction and single-strand breaks.
• The double-strand break model of recombination is also utilized.

The Holliday Model of Homologous Recombination

• First, homologous chromosomes align and single-strand breaks occur in the same position on both DNA molecules.
• A free end of each broken strand joins to the broken end of the other DNA molecule, creating a Holliday junction, and begins to displace the original complementary strand.
• Branch migration takes place as the two nucleotide strands exchange positions, creating the two duplex molecules.
• This view of the structure shows the ends of the two inter-connected duplexes pulled away from each other, and is rotated.
• Cleavage and rejoining, of the nucleotide strands produces noncrossover recombinants consisting of two heteroduplex molecules
• Noncrossover or crossover recombinant DNA, depending on whether cleavage is in the horizontal or the vertical plane.

Double-Strand Break Model of Recombination

• 2 double-stranded DNA molecules from homologous chromosomes align, before a double-strand break occurs in one of the molecules.
• Nucleotides are enzymatically removed, producing single-stranded DNA on each side, before a free 3' end invades and displaces a strand of the unbroken DNA molecule.
• The 3' end then elongates, further displacing the original strand
• The displaced strand forms a loop that base pairs with the broken DNA molecule, and DNA replicates on the bottom strand.
• The resulting strand attachment produces two Holliday junctions.