DNA Replication, Repair, and Recombination DNA
Replication
❖Base-Pairing Enables DNA Replication
➢Each strand is a template for a complementary strand
➢DNA replication is carried out by a cluster of proteins that form a “replication machine”
➢Semiconservative: each of the two new helix has one old strand and one new
❖DNA Synthesis Begins at Replication Origins
➢Started by “initiator proteins” that bind to specific DNA sequences called replication
origins
▪ Initiator proteins will unzip the DNA helix from the replication origin
➢A-T rich stretches of DNA are generally found at replication origins (weaker hydrogen
bonds)
➢Beginning DNA replication at many replication origins will shorten replication duration
❖Two Replication Forks Form at Each Replication Origin
➢Replication forks: Y-shaped junction of DNA molecules in the process of being replicated
▪ Two replication forks are formed at each replication origin
▪ The two forks move away from each other at the origin as the DNA is
unzipped and replicated
• DNA replication is termed “bidirectional” as a result
• Fork movement is very fast (hundreds to thousands of nucleotides/second)
❖DNA Polymerase Synthesizes DNA Using a Parental Strand as Template
➢DNA polymerase: An enzyme. Drives the movement of the replication fork.
Heart of the replication machine
➢Polymerization reaction involves the formation of a phosphodiester bond between the 3’
end of the growing DNA chain and the 5’phosphate group of the incoming nucleotide
➢Reaction is fueled by deoxyribonucleoside triphosphate
➢DNA polymerase does not dissociate from the DNA each time it adds a new
nucleotide. It stays attached
❖The Replication Fork is Asymmetrical
➢DNA is anti-parallel, so the two strands run in opposite directions
▪ One strand is synthesized from the 5’-to-3’ direction and the other is in the 3’-to-5’
direction
➢DNA polymerase only adds new subunits to the 3’ end of the DNA strand
▪ New DNA chains can only be made in the 5’-to-3’ direction
▪ Leading strand: does not require backstitching to make
➢3’-to-5’ strand is made discontinuously using “backstitching” (made in small separate
pieces and then joined together)
▪ Okazaki fragments: small pieces of DNA meant to form a continuous strand
▪ Lagging strand: backstitching causes a slight delay to its synthesis
➢Leading and lagging strands apply to all cells (prokaryotic and eukaryotic)
❖DNA Polymerase Is Self-correcting
➢The enzyme carefully monitors the base-pairing between each incoming nucleotide
and the template strand. It only catalyzes correct nucleotides.
➢It can correct errors through proofreading
▪ Polymerase clips off the misplaced nucleotide and tries again
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▪ Polymerization and proofreading are tightly coordinated and carried out by
different catalytic domains of the same polymerase molecule
▪ Synthesizing in the 3’-to-5’ direction will mean that proofreading is impossible
❖Short Lengths of RNA Act as Primers for DNA Synthesis
➢Primase: Base pairs RNA to the 3’ end of DNA and acts as a starting point for DNA
polymerase
▪ An example of RNA polymerase: enzyme that synthesizes RNA using DNA as a
template
▪ RNA contains uracil (U) instead of thymine
➢Leading strand: RNA primer only needed to start replication at the replication origin
➢Lagging strand: New primers are needed to keep polymerization going at each Okazaki
fragment
▪ Three additional enzymes are needed to join the fragments
• Act to remove the RNA primer and replace it with DNA and join the DNA
fragments together
• Nuclease degrades RNA primer
• Repair polymerase replaces RNA with DNA
• DNA ligase joins the 5’-phosphate end to the adjacent 3’-hydroxyl end
➢DNA polymerase replaces all RNA from the primers (RNA primers frequently contain
mistakes)
❖Proteins at a Replication Fork Cooperate to Form a Replication Machine
➢Two types of replication proteins
▪ DNA helicases
• Sits at the front of the replication machine. Uses ATP to propel forward and pry
apart the double helix
▪ Single-strand DNA-binding proteins
• Clings on single stranded DNA preventing them from re-forming base pairs
➢DNA topoisomerase: relieves tension in the DNA helix to allow for efficient
unwinding by DNA helicase
➢Sliding clamp: keeps DNA polymerase firmly attached to the template while it is
synthesizing new strands of DNA
➢Clamp loader: Uses ATP to lock a sliding clamp around a newly formed DNA double helix
▪ Occurs once on the leading strand and once for each Okazaki fragment on the lagging
strand
❖Telomerase Replicates the Ends of Eukaryotic Chromosomes
➢The lagging strand cannot be replicated at the end of a chromosome
➢Telomeres: long, repetitive nucleotide sequences at the ends of their chromosomes
➢Telomerase: telomere sequence attracts this enzyme. Extends the ends of the replicating
lagging strand by adding multiple copies of the same short DNA sequence to the
template strand.
▪ Allows for the lagging strand to be completed by conventional DNA replication
▪ Marks the true end of a chromosome. Not the end of fragments that accidentally
occur DNA Repair
❖DNA Damage Occurs Continually in Cells
➢DNA is continually undergoing thermal collision with other molecules, resulting
in major chemical changes
▪ Depurination
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➢Deamination: spontaneous loss of an amino group from a cytosine in DNA (produces
uracil)
➢Some DNA damage can stall DNA replication machinery at the site of the damage
❖Cells Possess a Variety of mechanisms for Repairing DNA
➢Information can be retrieved as long as one strand is undamaged
➢Basic pathway for repairing damaged DNA
▪ Damaged DNA is recognized and removed
▪ Repair DNA polymerase binds to the 3’ end and fills the gap
▪ DNA ligase seals the helix (same enzyme that joins Okazaki fragments)
❖A DNA Mismatch Repair System Removes Replication Errors That Escape Proofreading
➢Backup repair system: mismatch repair
▪ Mismatch: a mispaired nucleotide (will cause mutations)
▪ Mismatch repair proteins recognize the mismatch and removes the portion to rebuild it
• Only works on newly made strand to prevent changing a correct strand
➢Mismatch repair plays an important role in preventing
cancer
❖Double-Strand DNA Breaks Require a Different Strategy for
Repair
➢Dangerous because they can lead to fragmentation of chromosomes and loss of genes
➢Two strategies:
▪ Nonhomologous end joining
• Rapidly sticking the broken ends back together before DNA fragments drift apart
and get lost
• Can be imperfect and cause serious issues
▪ Homologous recombination
❖Homologous Recombination Can Flawlessly Repair DNA Double-Strand Breaks
➢If double-stranded breaks occur after replication, the unbroken strand can be used as a
template
➢Flawless repair: no loss of genetic information
➢Possibly the best DNA damage repair
❖Failure to Repair DNA Damage Can Have Severe Consequences for a Cell or Organism
➢A single nucleotide mistake can cause a deadly mutation
▪ Proteins do not from properly from an incorrect amino acid sequence
▪ Sickle-cell anemia
➢A mutation in the germ cell will be passed on to all cells in the body of the multicellular
organism that develops from it
➢Somatic cells (non-germ cells) need protection against mutation as well
▪ Cancer is an example of a mutation in somatic cells
❖A Record of the Fidelity of DNA Replication and Repair is Preserved in Genome Sequences
➢The majority of mutations do neither harm nor good
➢Favorable changes will tend to persist and spread and unfavorable changes will die out
▪ Natural selection
➢DNA between many species are vastly the same over the past 100 million years due
to DNA replication and repair
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