DNA Replication

• The goal of the Cell Cycle is to divide the cell
• Each cell has its own precisely timed cycle to determine when(or if) it’ll divide
• For cells that are actively dividing, there are 2 distinct phases:

  1. Interphase (cell prepares to divide)2. M phase (cell divides)
• During interphase, a “parent” cell grows to at least twice its size (so when splits in ½ it makes two normal size daughter cells)
• Interphase involves:

  • Increasing volume of cytoplasm
  • Synthesizing new/more proteins
  • Producing more organelles
  • Building more plasma membrane to fit around all the stuff listed above
  • Above all else: faithfully replicate the genome (DNA)!
• Interphase has 3 sub- phases

  1. G1 phase
  2. (cell grows in size and preps for S phase)
  3. S phase
  4. (cell replicates its genetic info in prep for dividing in M phase)
  5. G2 phase
  6. (more growth, final prep for M phase)
• During replication, the two strands of the original (parental) double helix separate and each parental strand serves as a template for synthesis of complementary “daughter” strands
• Some rules of DNA Replication:

  • New DNA synthesis occurs 5’ to 3’
  • Synthesis occurs via complementary base pairing
  • Replication occurs simultaneously on both strands
  • Catalyzed by DNA polymerase
• Semi-conservative: Each newly synthesized DNA molecule is composed of one original strand and one newly synthesized strand
• DNA replication: The process

  • Unwinding the helix
  • Helicase unwinds parental DNA double helix
  • Single-stranded binding proteins bind each of the parental DNA strands and prevent them from coming back together
• DNA replication: The process

  • Unwinding the helix
  • Topoisomerase relieves the stress of unwinding the DNA double helix
• DNA replication: The process

  1. Initiating Synthesis
  2. To get synthesis started RNA primase lays down RNA primer on each strand
• DNA replication: The process

  • Elongation
  • DNA polymerase extends RNA primer on each strand to make complementary daughter strands
• Why do we need an RNA primer?

  • To get synthesis started RNA primase lays down RNA primer on each strand
  • DNA polymerase extends RNA primer on each strand to make complementary daughter strand
• DNA Polymerase can’t initiate synthesis on its own!

  • DNA polymerase can only elongate the 3’ end of an existing nucleic acid (whether DNA or RNA)
• To get new DNA synthesis started, RNA primase will make a short piece of RNA complementary to the DNA template
• DNA polymerase will then build off RNA primer to elongate the new daughter strand
• The main site of action is the Replication Fork

  • Helicase is continuously unzipping the double helix to open up more template on each parent strand
• The main site of action is the Replication Fork

  • This creates a constantly progressing “fork” between double helix and unzipped strands
• The main site of action is the Replication Fork

  • On each strand, DNA polymerase operates at the fork to replicate more template as it becomes available (i.e., as DNA double helix is being unzipped)
• Complementary daughter strands are not synthesized the same way on both DNA templates

  • One daughter strand can be synthesized continuously
  • This is the leading strand
• Things we know about DNA:

  • DNA double helix is antiparallel
• Things we know about DNA:

  • During replication, both parent strands serve as templates for the synthesis of complementary daughter strands at the same time
• Things we know about DNA:

  • DNA can only be synthesized in 5’ to 3’ direction (in other words, new nucleotides can only be added to the 3’ end of previous nucleotides in the strand)
• Leading Strand

  • has its 3’ end pointing toward the replication fork; it is synthesized as one long, continuous molecule as the parental double helix is unwound
• Complementary daughter strands are not synthesized the same way on both DNA templates

  • But the other daughter strand must be synthesized in pieces (discontinuously)
  • Lagging strand
• Lagging strand

  • has its 3’ end pointed away from the replication fork, it must be synthesized in short, discontinuous (Fragmented) pieces
  • (the “pieces” of lagging strand DNA are called Okazaki fragments)
• What this means for the lagging strand...

  • As DNA unzips at replication fork, new RNA primer needs to keep being laid down to get DNA synthesis going again
  • When the growing Okazaki fragment comes into contact with the RNA primer of previous fragment a different DNA polymerase complex removes RNA primer and replaces it with DNA nucleotides
• When the replacement is complete, an enzyme called DNA ligase “glues” (ligates) discontinuous fragments together
• What feature of double-stranded DNA makes it necessary to have a leading strand and a lagging strand during replication?

  • the antiparallel orientation of the strand
• There are billions of nucleotides in the eukaryotic genome to replicate

  • Replication typically occurs only at a rate of ~ 50 nucleotides replicated per sec
• This means that a single daughter strand will be the leading strand at one replication fork and the lagging strand at the opposite replication fork
• Sometimes, DNA polymerase makes a mistake...

  • AMAZING feature of DNA polymerase – it can proofread!!!
  • In other words, DNA polymerase can detect when it has mismatched a nucleotide base