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Chapter 11

Notes Chapter 11 (Lectures 4, 7) - Biochem 2B03.docx

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Margaret Fahnestock

Biochem 2B03 Jasmyn Lee Chapter 11: Structure of Nucleic Acid 11.1 How Do Scientists Determine The Primary Structure of Nucleic Acids? The nucleotide sequence of DNA can be determined from the electrophoretic migration of a defined set of polynucleotide fragments  Chain termination/dideoxy method – more common protocol for nucleic acid sequencing o Fredrick Sanger o Relies on enzymatic replication of the DNA to be sequenced Sanger’s chain termination/dideoxy method uses DNA replication to generate a defined set of polynucleotide fragments  Biochemistry of DNA Replication o DNA polymerase copies the sequence of nucleotides in one strand in a complementary fashion to form a new second strand o Each original strand of the double helix serves as a template o DNA polymerase carries this out in the presence of four deoxynucleotide monomers and copies single- stranded DNA, provided a double-stranded region of DNA is artificially generated by adding a primer  Primer – an oligonucleotide capable of forming a short stretch of dsDNA by base pairing with the ssDNA o Primer must have a free 3’-OH end from which the new polynucleotide chain can grow as the first residue is added in the initial step of the polymerization process o DNA polymerase synthesize new strands by adding successive nucleotides in the 5’3’ direction  Chain Termination Protocol – Sanger DNA sequence method o Clones derived from PCR products need to be sequenced to rule out errors o Sanger sequencing makes use of a short DNA primer and a single subunit DNA polymerase (derived from T7 bacteriophage usually o Primer requirement is met by an appropriate oligonucleotide o Reaction run in the presence of  All four deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) which are the substrate for DNA Polymerase  All four corresponding 2’,3’-dideoxynucleotides o Depends on base-specific inhibitors o 2’,3’-dideoxy nucleoside triphosphate lack 3’-OH groups and cannot serve as acceptors for 5’-nucleotide addition in the polymerization reaction – inhibits progression of DNA polymerase; chain is terminated where they are incorporated 1 Biochem 2B03 Jasmyn Lee  No nucleophile to attach the 5’ phosphate o Fewer dideoxynucleotide than deoxynucleotides – chain is terminated infrequently and randomly – a population of new strands of varying lengths is synthesized o Add fluorescently labeled 2’3’ dideoxynucleoside triphosphates to a DNA synthesis reaction at ~10x lower concentration than the 2’ deoxynucleoside triphosphates 1. Causes chain termination 2. Labels the terminated ends with a base-specific fluorescent label o These can then be separated on the basis of length using capillary gel electrophoresis o The order of different colored fluorescent markers allows you to read the DNA sequence directly o Eg/ Gel read 5’TTGTCGAAGTCAG3’  sequence is complementary to template DNA strand 5’CTGACTTCGACAA3’ 11.2 What sorts of Secondary Structures Can Double-Stranded DNA Molecules Adapt?  DNA usually occurs in the form of double stranded molecules o DNA is a two chain structure with hydrogen bonds formed between opposing based on the two antiparallel chains o Polar sugar phosphate backbones on the outside; bases stacked on the inside (0.6 nm apart when in ladder-like structure) o Ladder-like structure converts into a double helix when given a right handed twist – brings base pair rungs closer together (0.34 nm apart)  B-DNA  Watson-Crick base pairs (A:T pair and G:C pair) have virtually identical dimensions  DNA double helix is a stable structure o H-Bonds  2 in A:T pair  3 in G:C pair  H bonds form between H 2 and bases when strands are separated 2 Biochem 2B03 Jasmyn Lee  Polar atoms in sugar-phosphate backbone form external H bonds with2H O o Electrostatic Interactions  Negatively charged phosphate groups along the sugar-phosphate backbone – keep strands apart except when base pairing occurs  Charges become electrostatically shielded from one another because divalent cations (i.e. Mg ) bind strongly to the anionic phosphates o Van der Waals and Hydrophobic Interactions  Base pairs stick together through π,π-electronic interactions and hydrophobic forces  Two glycosidic bonds holding the bases in each base pair are not directly across the helix from each other  Sugar-phosphate backbones of the helix are nonequally spaced along the helix axis and the grooved between them are not the same size  major groove and minor groove o The “tops” of the bases line the “floor of the major groove”  Top defined as placing the glycosidic bond at the bottom o The “bottoms” of the bases line the floor of the minor groove  Some proteins that bind to DNA can recognize specific nucleotide sequences by reading the pattern of H-bonding possibilities presented by the edges of the bases in these grooves  Watson-Crick base pairs as they project into the grooves o Chemical information in major groove: CG base paiNH 2 O GC base pair O NH 2 AT base pair NH 2 CH 3 TA base pair CH 3 NH 2 o Chemical information in minor groove: GC base paiNH 2 O CG base pair O NH 2 AT base pair O TA base pair O 3 Biochem 2B03 Jasmyn Lee  Double helical structures can adopt a number of stable conformations o Helical Twist – rotation around the axis of the double helix of one base pair relative to the next o Propeller Twist – involves rotation around a different axis (namely an axis perpendicular to the helix axis)  A-Form DNA is an alternative form of right-handed DNA  Z-DNA is a conformational variation in the form of a left handed double helix Three kinds of DNA  A form DNA – right-handed, short and broad, 2.3 Å/bp, 11 bp per turn  B form DNA
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