DNA Replication and Repair

• DNA replication is semi-conservative

  1. each parent DNA strand is a template for a new complementary, antiparallel daughter strand
  2. each daughter duplex strand contains:
  3. one strand from the parent
  4. one newly synthesized strand (antiparallel, complementary)
  5. two parent strands are separated forever
  6. two new duplex strands are identical to parent duplex
• DNA polymerases only synthesize DNA in the 5' to 3' direction
• parental strands are antiparallel
• what is needed to replicate DNA?

  • single-strand DNA binding protein to prevent reannealing
  • DNA ligase to seal nicks
  • a clamp to enhance Pol processivity
  • a clamp loader to load the clamp
  • DNA polymerase(s) to copy the template
  • DNA helicase to unwind duplex DNA
  • primase to initiate de novo synthesis
  • a nuclease to remove RNA primers
• unwinding DNA at the replication fork

  • helicase uses energy from ATP hydrolysis to separate strands
  • this generates single stranded DNA and torsional strain (relieved by SSB (single-strand binding proteins) and topoisomerases)
• DNA polymerases of the bacteria and eukaryotes

  • bacteria have 5
  • yeast have 6
  • humans have 15
• e. coli and gram-negative bacteria polymerases

  • Pol I: removal of primers (5' nuclease) and maturation of okazaki fragments (lagging strand) and DNA repair
  • Pol II: translesion DNA repair (damage bypass)
  • Pol III: main replication of polymerase (processive)
  • others: translesion and other DNA repair synthesis
• yeast and other eukaryotes polymerases

  • Pol alpha: priming and first DNA synthesis (DNA replication)
  • Pol delta: lagging strand synthesis (DNA replication)
  • Pol epsilon: leading strand synthesis (DNA replication)
  • Pol beta: DNA repair
  • Pol gamma: mitochondrial DNA replication
  • others: translesion repair (damage bypass)
• DNA polymerases require a primer, a template, all 4 dNTPs and Mg++

  • RNA polymerases carry out a similar reaction, require ribo-NTPs (nucleotide triphosphate) and do not require a primer to initiate synthesis of RNA
• some DNA polymerases have 3' to 5' proofreading

  • base-base mismatch causes DNA synthesis to slow
  • 3' primer frays and translocates to exo active site
  • release of single dNMPs (nucleotide monophosphate), including mispaired dNMP
• a typical DNA (bacterial) replisome

  • the clamp loader loads the sliding clamp onto the DNA pol and the DNA primer-template junction
  • removal or RNA primers requires RNase H (prokaryotes) or FEN-1 (eukaryotes)
  • coupled leading and lagging strand DNA synthesis at a replication fork
• helicase

  • unwinds DNA duplex
  • allows access by remainder of replication complex
• ssDNA binding protein (tan)

  • maintains fork stability
  • prevents ssDNA degradation
• primase

  • adds short RNA primer complementary to ssDNA templates
• DNA polymerase alpha

  synthesizes short stretch of DNA from RNA primer on lagging strand (beginning of lagging okazaki fragment)
• DNA polymerase epsilon and delta

  • extends where alpha polymerase leaves off
  • more accurate and processive (PCNA)
  • leading strand synthesis
• RNaseH and Fen1 remove RNA primers
• DNA ligase forms 3' to 5' phosphodiester bonds connecting two DNA fragments
• RNase H, DNA pol I and DNA ligase cooperate to remove RNA primers and seal nicks left by DNA synthesis
• linear chromosomes create "end replication" problem

  • ends are shortened at each round of replication
  • telomeres help solve this problem
  • strands of repeating sequences (T/A/G)
  • telomerase adds theses sequences to the ends of the strands
• when does replication occur (prokaryotes)?

  • can occur at any time
  • can start prior to previous replication completion
  • about 40 minutes for 4-6 xx 10^6 bp
• when does replication occur (eukaryotes)?

  • highly regulated in cell cycle (S phase)
  • once and only once per S phase
  • about 8 hours for human cells in culture
• DDI (older) and emtricitibine (newer) are inhibitors of DNA synthesis by reverse transcriptase. they are commonly used as anti-HIV drugs
• acyclovir and gancyclovire are "nucleoside analogues" of guanosine and inhibitors of DNA synthesis by herpes viruses. the compounds are phosphorylated by a viral kinase and the resulting nucleotides inhibit DNA synthesis by the viral and cellular DNA polymerases, thus only infected cells are inhibited
• DNA damage

  • cells are constantly exposed to agents the can cause damage to their genome (hydrolysis, reactive oxygen species, UV light, alkylating agents)
  • unrepaired DNA damage can lead to mutagenesis and potentially tumorigenesis or cell death
• intrinsic or spontaneous DNA damage

  • spontaneous base loss
  • (uncorrected) replication errors
• exogenous DNA damage

  • ionizing radiation (x-rays)
  • ultraviolet light (thymine dimers)
  • environmental chemicals
• endogenous DNA damage

  • reactive oxygen (H2O2, O2-)
• base alterations can lead to mutations

  • oxidation
  • alkylation
  • cross-links
  • large base adducts
• exogenous damage: exposure to ultraviolet radiation

  • exposure to UV light introduces pyrimidine dimers in DNA (primarily T-T). these lesions cause distortions in the DNA structure that are recognized by repair enzymes. unrepaired pyrimidine dimers are potentially lethal or mutagenic
  • geometric distortions affect recognition, repair and toxicity of lesion
• 3 types of excision repair

  1. base excision repair
  2. nucleotide excision repair
  3. mismatch excision repair
• base excision repair


  excision of the single errant NT (eg. uracil, oxidized base)

  • many different DNA glycosylases
  • specific to recognize particular DNA
  • lyase/disterase of 5' endonuclease required to break the sugar/phosphate backbone
  • DNA Pol beta for majority of resynthesis
  • excised region is about 1 nucleotide long (sometimes up to 5-7)
  • repair of bulky lesions
• nucleotide excision repair


  various bulky, helix-distorting lesions (particularly pyrimidine dimers), excision of a patch (oligonucleotide) surrounding lesion

  • repair of helix distorting lesions, particularly CPDs and 6-4 photoproducts
  • XPC
  • TFIIH
  • RPA
  • XPF and XPG
  • RFC
  • PCNA
  • DNA Pol delta/epsilon
  • excised region is about 27 nucleotides long
• mismatch excision repair 


  errors arising from DNA replication, excision of an extended region (up to 1kb) surrounding the lesion
• • XPC = lesion recognition and further DNA bending
• • TFIIH = transcription factor IIH complex (10 subunits, two of which are XPB and XPD)
• • RPA = replication protein A, a single stranded DNA binding protein
• • XPF and XPG = endonucleases that work with helicases (XPD and XPB) to remove the lesions
• • RFC = replication factor C, the clamp loader
• • RFC = replication factor C, the clamp loader