LC 04: DNA Replication

• Steps in bacterial DNA replication:

  1. Origin of replication

  2. The Binding of initiator proteins

  3. Unwinding by DNA helicase

  4. The Binding of single strand binding proteins

  5. RNA primers made by primase

  6. DNA polymerase sliding clamps

  7. Nick sealing
• Initiator proteins bind to the origin of replication, helping helicase bind; this process requires ATP
• Helicase loading protein assists DNA helicase in binding to DNA; helicase unwinds and separates the DNA strands using ATP
• Helicases move in the 5' to 3' direction along the lagging strand template, separating the DNA double helix like a zipper
• Single strand binding proteins prevent DNA strands from reannealing after helicase action and keep single strands separate; they are present on both leading and lagging strand templates
• RNA primers, synthesized by primase, provide a starting point for DNA polymerase to add nucleotides in the 5' to 3' direction
• DNA primase synthesizes RNA from 5' to 3' and reads from 3' to 5', adding on to a 3' end
• The primer made by primase is RNA, serving as a temporary starting point for DNA polymerase to extend onto
• The RNA primer is temporary, inaccurate, and slow, allowing DNA polymerase to start adding DNA and later replace the RNA
• Primase, DNA primase, and RNA primase are different names for the same enzyme that makes an RNA primer for DNA polymerase to add onto
• DNA polymerase adds nucleotides from 5' to 3' on the new strand, starting from a 3' end and reading the template from 3' to 5'
• The sliding clamp helps keep DNA polymerase attached to the DNA template during replication, preventing it from floating away after adding a few nucleotides
• Both the leading and lagging strand templates have single-stranded DNA binding proteins to assist in replication
• The helicase moves in the 5' to 3' direction, separating the strands, while the primase makes the primer in the opposite direction, causing a bit of DNA looping for efficient replication
• The primosome consists of the primase and helicase working together to initiate DNA replication
• DNA polymerase adds deoxyribonucleoside triphosphates to the 3' end of the new strand, cutting off two phosphate groups and extending the DNA chain in the 5' to 3' direction
• DNA replication involves the following steps:

  • Helicase separates the parental DNA double helix
  • The lagging strand template is bound to a primase
  • Newly synthesized strand on the leading strand
  • Sliding clamp is needed on the leading strand
  • DNA polymerase is required on both strands
  • Single-stranded binding proteins are typically present
  • Lagging strand also requires single-stranded binding proteins and a sliding clamp
• Okazaki fragments on the lagging strand are linked together by:

  • Primase laying down a primer
  • DNA polymerase adding nucleotides to the 3' end of the new RNA primer to synthesize the Okazaki fragment
  • Nucleases removing the RNA primer
  • DNA polymerase filling in the gap left by the removed RNA primer
  • DNA ligase sealing the gap between the 5' and 3' ends
• For the leading strand, the RNA primer is easily replaced because before it, there is a 3' end of a lagging strand
• Nick sealing by DNA ligase involves using ATP to catalyze the reaction and form a covalent bond between the sections
• Telomeres are involved in aging: before a cell divides, it replicates its DNA but replication is not complete at the ends, causing telomeres to get shorter with each replication
• Proteins detect when telomeres get too short, signaling the cell to stop dividing, which can lead to the inability to replace damaged or old cells, contributing to aging
• DNA replication:

  • DNA ends get shorter with each replication
  • Proteins detect when the ends get too short and signal the cell to stop dividing to prevent loss of genetic material
• In cancer, cells do not stop dividing, leading to uncontrolled cell division
• Telomeres are like the protector ends of DNA double helix, preventing loss of genetic material during cell division
• DNA unwinding during replication causes supercoiling and torsional strain, solved by enzymes called Topoisomerases that create temporary single-strand breaks to relieve tension
• In DNA replication, the leading strand is continuously elongated by DNA polymerase from the 3' end, while the lagging strand faces challenges due to primase not efficiently placing a primer at the very end
• The shortening of the 5' end of the daughter DNA strand in DNA replication, particularly on the lagging strand, can lead to the loss of sequence information, affecting future replication processes
• Tas enzyme plays a crucial role in DNA replication by adding repetitive sequences to the 3' end of the parental strand, specifically on the lagging strand, using an RNA template to extend the DNA strand
• The ends of chromosomes contain a g-rich series of repeats called Aamir Tas
• Toras elongates the parental strand in the 5' to 3' direction and adds additional repeats as it moves down the parental strand
• The lagging strand is then completed by DNA polymerase Alpha, which carries a DNA primase as one of its subunits
• The original information at the ends of linear chromosomes is completely copied in the new DNA
• In DNA replication, an RNA primer is needed to make the strand longer, eventually requiring DNA polymerase to add on to that primer
• DNA ligase seals the nick after the primer is removed
• Tas is abundant in stem and germline cells but not in somatic cells
• Most cancer cells produce high levels of Tas as they never stop replicating, leading to tumor formation
• DNA replication has high fidelity, with DNA polymerases having an error rate of about 1 in 10 to the 9 nucleotides inserted
• To maintain accuracy in DNA replication, mechanisms like 3' to 5' exonuclease repair and strand-directed mismatch repair are employed
• 3' to 5' exonuclease repair acts like a backspace mechanism, cutting off misincorporated nucleotides