lecture 9

DNA Repair

• DNA can be damaged by environmental factors
• Most DNA damage is repaired by excision of the damaged DNA
  • The damaged DNA is removed
  • The resulting gap is filled in with newly synthesized DNA
  • The undamaged, complimentary strand is used as a template
• Mismatch repair specifically removes mismatched bases from newly synthesized DNA
• Mutations in genes that code for DNA repair proteins are frequently found in cancer cells
• Double-strand breaks can be repaired by nonhomologous end joining (NHEJ) or homologous recombination (HDR)
  • NHEJ is error-prone
  • HDR is more accurate and uses undamaged homologous DNA as a template
• Excision repair mechanisms remove damaged DNA in several steps:
  • Damaged DNA is recognized by specific enzymes
  • Damaged DNA is excised
  • The gap is filled in by repair DNA polymerase
    • The undamaged complimentary strand is used as a template
  • The backbone is sealed with DNA ligase
• Base-excision repair corrects damaged bases
• Nucleotide-excision repair corrects damage that distorts the double helix
• Mismatch repair corrects mismatched bases
• Nonhomologous end joining repairs double-strand breaks by directly ligating the broken ends
• Homologous recombination repairs double-strand breaks using a homologous DNA sequence as a template

Molecular Cloning & DNA Sequencing

• Restriction endonucleases cleave specific DNA sequences yielding defined fragments of DNA molecules
• DNA fragments can be ligated into a plasmid vector which is then replicated in an appropriate host cell and isolated as molecular clones
• The nucleotide sequence of cloned DNA fragments can be determined using Sanger sequencing
• Molecular cloning is used for:
  • Characterizing and studying the function of genes
  • Characterizing and studying the function of genomic regulatory regions
  • Production of transgenic animal models of human diseases
  • Production of recombinant proteins
  • Transgenic plants and animals for agricultural, pharmaceutical, and nutritional enhancements
  • Gene therapy delivery
• The four main steps of molecular cloning are:
  • Digest DNA with restriction endonucleases
  • Ligate DNA fragments into plasmid vectors
  • Transform E. coli with recombinant DNA plasmids
  • Select and characterize the recombinant clones
• Restriction endonucleases cleave DNA at specific recognition sites of four to eight base pairs
• Restriction endonucleases can result in blunt or sticky ends
• The natural function of restriction endonucleases is to protect bacteria from foreign DNA
• Plasmid vectors are small circular DNA molecules that replicate independently of the host chromosome
• Molecular cloning begins with restriction endonuclease digestion of the genome of interest and the plasmid vector
• Ligation joins the digested genome fragments into the plasmid vector
• E. coli is transformed with the recombinant DNA plasmids
• Transformants are selected and characterized
• Sanger sequencing uses DNA polymerase to synthesize a complementary strand of DNA
  • Dideoxynucleosides are used to terminate DNA synthesis at specific bases
  • This produces fragments of different lengths that can be separated by electrophoresis
  • The sequence of the DNA fragment can be determined by analyzing the lengths of the fragments

This quiz covers the intricate processes involved in DNA repair, focusing on how damaged DNA is recognized, excised, and repaired by various mechanisms. You will explore excision repair, mismatch repair, and the roles of specific proteins in these pathways, especially in relation to cancer. Test your knowledge on error-prone and accurate repair systems as well.