CHAPTER 8

• DNA replication


  The process by which the genetic material is copied

  
• DNA replication


  • It occurs very quickly
  • It occurs very accurately
  • It occurs at the appropriate time in the life of the cell
• Bacterial chromosomal replication


  1. DNA synthesis begins at the origin of replication

  2. Synthesis proceeds bidirectionally around the bacterial chromosome

  3. The two replication forks eventually meet at the opposite side of the bacterial chromosome
• Origin of replication (oriC)


  The site where DNA replication begins in bacteria

  
• Functionally significant sequences in oriC

  • AT-rich region
  • DnaA boxes
  • GATC methylation sites
• Initiation of replication


  1. DnaA protein binds to DnaA boxes

  2. DnaA proteins form a large complex

  3. Other proteins like HU and IHF bind

  4. This causes separation of the AT-rich region

  
• DNA helicase


  • Composed of six subunits
  • Travels along the DNA in the 5' to 3' direction
  • Uses energy from ATP
  • Initiates bidirectional replication

  
• DNA replication process


  1. DNA helicase separates the DNA strands

  2. DNA gyrase alleviates positive supercoiling

  3. Single-strand binding proteins keep the strands apart

  4. RNA primers are synthesized by primase

  5. DNA polymerase III synthesizes the daughter strands

  6. DNA polymerase I removes RNA primers and fills gaps

  7. DNA ligase joins Okazaki fragments

  
• DNA polymerases


  Enzymes that catalyze the attachment of nucleotides to synthesize a new DNA strand

  
• DNA polymerases in E. coli

  • DNA pol I
  • DNA pol II
  • DNA pol III
  • DNA pol IV
  • DNA pol V

  
• DNA pol I


  Removes RNA primers and replaces them with DNA
• DNA pol III


  • Responsible for most DNA replication
  • Composed of 10 different subunits

  
• DNA polymerase III


  • The α subunit catalyzes bond formation between nucleotides
  • The other 9 subunits fulfill other functions
  • The complex of all 10 subunits is the DNA pol III holoenzyme

  
• Leading strand


  One RNA primer is made at the origin, DNA pol III attaches nucleotides in a 5' to 3' direction

  
• Lagging strand


  Many RNA primers are required, DNA pol III synthesizes small DNA fragments (Okazaki fragments)
• Completion of DNA replication

  1. DNA pol I removes RNA primers and fills gaps with DNA

  2. DNA ligase forms phosphodiester bonds to connect Okazaki fragments

  
• DNA polymerase reaction


  Forms a covalent ester bond between the innermost phosphate of the incoming nucleotide and the 3'-OH of the previous nucleotide, releasing pyrophosphate
• DNA replication complexes


  • The primosome is the complex of DNA helicase and primase
  • The replisome is the complex of the primosome and two DNA pol III holoenzymes

  
• Dimeric DNA polymerase


  Two DNA pol III proteins act in concert to replicate both the leading and lagging strands

  
• Lagging strand synthesis


  1. The lagging strand is looped to allow the polymerase to synthesize Okazaki fragments in the 5' to 3' direction

  2. Upon completion of an Okazaki fragment, the polymerase is reloaded at the next RNA primer

  
• Termination sequences (ter)


  Sequences that stop the movement of replication forks
• Tus protein


  Binds to ter sequences and stops the advancement of replication forks
• 5' to 3' direction

  1. Polymerase synthesizing the lagging strand is moving toward the replication fork

  2. Upon completion of an Okazaki fragment, the enzyme releases the lagging template strand

  3. Clamp loader complex reloads the polymerase at the next RNA primer

  4. Another loop is then formed

  5. This process is repeated over and over again

  
• DNA Replication Complexes


  • On the opposite side of the chromosome to oriC is a pair of termination sequences called ter sequences
  • T1 stops counterclockwise forks, T2 stops clockwise forks
  • The protein tus (termination utilization substance) binds to the ter sequences
  • tus bound to the ter sequences stops the movement of the replication forks
• Termination of Replication


  1. DNA replication ends when oppositely advancing forks meet (usually at T1 or T2)

  2. DNA ligase covalently links the two daughter strands

  3. DNA replication often results in two intertwined molecules

  4. Intertwined circular molecules are termed catenanes

  5. These are separated by the action of topoisomerase

  
• DNA replication fidelity


  • Mistakes during the process are extremely rare
  • DNA pol III makes only one mistake per 10^8 bases made
  • Instability of mismatched pairs
  • Configuration of the DNA polymerase active site
  • Proofreading function of DNA polymerase

  
• Proofreading Mechanisms


  1. Instability of mismatched pairs

  2. Configuration of the DNA polymerase active site

  3. Proofreading function of DNA polymerase

  
• Bacterial cells can divide into two daughter cells at an amazing rate
• It is critical that DNA replication take place only when a cell is about to divide
• Bacterial cells regulate the DNA replication process by controlling the initiation of replication at oriC

  
• Eukaryotic DNA replication


  • Not as well understood as bacterial replication
  • Similarities with bacterial replication
  • More complex due to large linear chromosomes, tightly packed chromatin, and more complicated cell cycle regulation

  
• Origins of replication in Saccharomyces cerevisiae

  • Termed ARS elements (Autonomously Replicating Sequence)
  • About 50 bp in length
  • High percentage of A and T
  • ARS consensus sequence (ACS) ATTTAT(A or G)TTTA

  
• Multiple Origins of Replication

  1. Replication begins with assembly of the prereplication complex (preRC)

  2. An important part of the preRC is the Origin recognition complex (ORC)

  3. Binding of MCM completes DNA replication licensing

  4. At least 22 additional proteins are required to initiate synthesis during S phase

  
• Eukaryotic DNA Polymerases


  • Alpha (α), delta (δ), epsilon (ε) and gamma (γ) have the primary function of replicating DNA
  • α, δ and ε → Nuclear DNA
  • γ → Mitochondrial DNA

  
• DNA pol α


  • The only polymerase to associate with primase
  • Synthesizes a short RNA-DNA hybrid primer
  • Exchange of DNA pol α for δ or ε is required for elongation of the leading and lagging strands
• DNA pol ζ


  Not involved in DNA replication, plays a role in base-excision repair

  
• Lesion-replicating polymerases


  • Involved in the replication of damaged DNA
  • Can synthesize a complementary strand over the abnormal region

  
• Flap Endonuclease Removes RNA Primers

  1. Polymerase δ runs into primer of adjacent Okazaki fragment

  2. Pushes portion of primer into short flap

  3. Flap endonuclease removes the primer

  4. Long flaps are removed by Dna2 nuclease/helicase
• Telomeres


  • Linear eukaryotic chromosomes have telomeres at both ends
  • Telomere refers to the complex of telomeric DNA sequences and bound proteins
• DNA polymerases


  • Synthesize DNA only in the 5' to 3' direction
  • Cannot initiate DNA synthesis