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DNA-binding Domains Structural considerations of the DNA double helix PowerPoint Presentation
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DNA-binding Domains Structural considerations of the DNA double helix

DNA-binding Domains Structural considerations of the DNA double helix

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DNA-binding Domains Structural considerations of the DNA double helix

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  1. DNA-binding Domains • Structural considerations of the DNA double helix • Families of DNA-binding proteins

  2. DNA-binding domains: Let’s look at DNA first

  3. Heparin can be used to purify DNA-binding proteins Virtually ALL DNA-binding proteins have intrinsic affinity to the phosphate backbone of DNA (i.e. they have a non-sequence specific affinity for DNA)

  4. The minor groove harbors little chemical Information G:C C:G A:T T:A

  5. Helix-Turn-Helix

  6. The Helix-Turn-Helix motif was reinvented multiple times Homeodomain (yeast MatA1) TEA domain (human TEF-1) Anbanandam A et al. PNAS 2006;103:17225-17230

  7. A (famous) mutation in a homeotic gene: Ultrabithorax Wild type Ultrabithorax From: Lawrence, The making of a fly

  8. Basic Region Leucine zipper (bZIP) Dimer of two large a-helices that form a coiled coil Examples: FOS and JUN

  9. Basic Region Helix-loop-Helix domain (bHLH) Example: MyoD

  10. Proto-oncogene c-myc is a bHLH-Zip transcription factor

  11. The C2H2 zinc finger domain Example: TFIIIA

  12. Nuclear Receptors bind DNA via a pair of C4 zinc fingers Four cysteines are complexed with a Zn++ ion

  13. Structural properties of zinc fingers binding to DNA C2H2 type C4 type (nuclear receptors)

  14. Dimerization domains often occur in combinations

  15. Experimental tools to define regulatory DNA elements Testing function: Mutations/Promoter bashing Testing binding: DNAse I footprinting EMSA: Electrophoretic Mobility Shift Assay chIP: chromatin Immunoprecipitation

  16. Defining promoter elements via promoter bashing ( ) * Expression Reporter Gene upstream region +++++ +++++ +++++ ++++ ++++ +++ + * + (+) - + +(+)

  17. DNAse I footprinting • Idea: DNA-bound protein protect • DNA from digestion • Radiolabel DNA fragment at one end • Incubate protein with DNA • Digest DNA with low concentration • of DNAse I • Run fragments on high resolution gel • Map location of “footprint” based on • size of fragments

  18. - Electrophoretic Mobility Shift Assay I + antibody + + + + labeled DNA + + + protein + “cold” competitor DNA +

  19. Electrophoretic Mobility Shift Assay II Cold specific competitor DNA: usually a fragment of DNA, always unlabeled, such as a double-stranded oligonucleotide that contains the suspected DNA recognition sequence or mutations thereof. Typically, 10-100fold molar excess is used. If higher amounts are needed, the complex in question is likely to be non-specific. Non-specific competitor DNA: Because every DNA-binding protein exhibits non-specific affinity for DNA, one needs to add high amounts of non-specific DNA to saturate this activity. This is particularly important when using nuclear extracts. Most often, one uses synthetic DNA, such as poly [d(I-C)]. DNA-binding proteins: can be added in the form of crude nuclear extracts, biochemically purified proteins or in-vitro translated proteins.