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Structure of Nucleic Acids: Sequencing and Secondary Structure

This chapter explores the primary structure of nucleic acids, including DNA sequencing methods such as the chain termination and chemical cleavage methods. It also discusses the secondary structure of DNA and the discovery of Z-DNA.

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Structure of Nucleic Acids: Sequencing and Secondary Structure

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  1. Chapter 12 Structure of Nucleic Acids to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida 32887-6777

  2. Outline • 12.1 Primary Structure of Nucleic Acids • 12.2 ABZs of DNA Secondary Structure • 12.3 Denaturation and Renaturation of DNA • 12.4 Tertiary Structure of DNA • 12.5 Chromosome Structure • 12.6 Chemical Synthesis of Nucleic Acids • 12.7 Secondary and Tertiary Structure of RNA

  3. Primary Structure Sequencing Nucleic Acids • Chain termination method (dideoxy method), developed by F. Sanger • Base-specific chemical cleavage, developed by Maxam and Gilbert • Both use autoradiography - X-ray film develops in response to presence of radioactive isotopes in nucleic acid molecules

  4. DNA Replication • DNA is a double-helical molecule • Each strand of the helix must be copied in complementary fashion by DNA polymerase • Each strand is a template for copying • DNA polymerase requires template and primer • Primer: an oligonucleotide that pairs with the end of the template molecule to form dsDNA • DNA polymerases add nucleotides in 5'-3' direction

  5. Chain Termination Method Based on DNA polymerase reaction • Run four separate reactions • Each reaction mixture contains dATP, dGTP, dCTP and dTTP, one of which is P-32-labelled • Each reaction also contains a small amount of one dideoxynucleotide: either ddATP, ddGTP, ddCTP or ddTTP

  6. Chain Termination Method • Most of the time, the polymerase uses normal nucleotides and DNA molecules grow normally • Occasionally, the polymerase uses a dideoxynucleotide, which adds to the chain and then prevents further growth in that molecule • Random insertion of dd-nucleotides leaves (optimally) at least a few chains terminated at every occurrence of a given nucleotide

  7. Chain Termination Method • Run each reaction mixture on electrophoresis gel • Short fragments go to bottom, long fragments on top • Read the "sequence" from bottom of gel to top • Convert this "sequence" to the complementary sequence • Now read from the other end and you have the sequence you wanted - read 5' to 3'

  8. Chemical Cleavage Method Not used as frequently as Sanger's • Start with ssDNA labelled with P-32 at one end • Strand is cleaved by chemical reagents • Assumption is that strands of all possible lengths, each cleaved at just one of the occurrences of a given base, will be produced. • Fragments are electrophoresed and sequence is read

  9. Chemical Cleavage Method Four reactions are used • G-specific cleavage with dimethyl sulfate, followed by strand scission with piperidine • G/A cleavage: depurination with mild acid, followed by piperidine • C/T cleavage: ring hydrolysis by hydrazine, followed by piperidine • C cleavage: same method (hydrazine and piperidine), but high salt protects T residues

  10. Chemical Cleavage Method Reading the gels... • It depends on which end of the ssDNA was radioactively labelled! • If the 5'-end was labelled, read the sequence from bottom of gel to top (5' to 3') • If the 3'-end was labelled, read the sequence from top of gel to bottom (5' to 3') • Note that the nucleotide closest to the P-32 will be missed in this procedure

  11. The ABZs of DNA Secondary Structure • See Figure 12.10 for details of DNA secondary structure • Sugar-phosphate backbone outside • Bases (hydrogen-bonded) inside • Right-twist closes the gaps between base pairs to 3.4 A (0.34 nm) in B-DNA

  12. The “canonical” base pairs See Figure 12.10 • The canonical A:T and G:C base pairs have nearly identical overall dimensions • A and T share two H-bonds • G and C share three H-bonds • G:C-rich regions of DNA are more stable • Polar atoms in the sugar-phosphate backbone also form H-bonds

  13. Major and minor grooves See Figures 12.10, 12.11 • The "tops" of the bases (as we draw them) line the "floor" of the major groove • The major groove is large enough to accommodate an alpha helix from a protein • Regulatory proteins (transcription factors) can recognize the pattern of bases and H-bonding possibilities in the major groove

  14. Comparison of A, B, Z DNA See Table 12.1 • A: right-handed, short and broad, 2.3 A, 11 bp per turn • B: right-handed, longer, thinner, 3.32 A, 10 bp per turn • Z: left-handed, longest, thinnest, 3.8 A, 12 bp per turn • See Figure 12.13

  15. Z-DNA Discovered by Alex Rich • Found in G:C-rich regions of DNA • G goes to syn conformation • C stays anti but whole C nucleoside (base and sugar) flips 180 degrees • Result is that G:C H-bonds can be preserved in the transition from B-form to Z-form!

  16. 12.3 Denaturation of DNA See Figure 12.17 • When DNA is heated to 80+ degrees Celsius, its UV absorbance increases by 30-40% • This hyperchromic shift reflects the unwinding of the DNA double helix • Stacked base pairs in native DNA absorb less light • When T is lowered, the absorbance drops, reflecting the re-establishment of stacking

  17. 12.4 Supercoils and Cruciforms • In duplex DNA, ten bp per turn of helix • Circular DNA sometimes has more or less than 10 bp per turn - a supercoiled state • Enzymes called topoisomerases or gyrases can introduce or remove supercoils • Cruciforms occur in palindromic regions of DNA • Negative supercoiling may promote cruciforms

  18. Chromosome Structure • Human DNA’s total length is ~2 meters! • This must be packaged into a nucleus that is about 5 micrometers in diameter • This represents a compression of more than 100,000! • It is made possible by wrapping the DNA around protein spools called nucleosomes and then packing these in helical filaments

  19. Nucleosome Structure • Chromatin, the nucleoprotein complex, consists of histones and nonhistone chromosomal proteins • Histone octamer structure has been solved (without DNA by Moudrianakis, and with DNA by Richmond) • Nonhistone proteins are regulators of gene expression

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