Introduction to DNA Structure and Function PDF

Module 2: DNA [email protected] Please sit with your PBL groups What to expect in Module 2 (and 3): o Problem-based questions (applied) o Understand the concepts and applications still have to learn basic facts, to be able to apply them o Understand the techniques (and how to apply...

Module 2: DNA [email protected] Please sit with your PBL groups What to expect in Module 2 (and 3): o Problem-based questions (applied) o Understand the concepts and applications still have to learn basic facts, to be able to apply them o Understand the techniques (and how to apply them) o Interpret data and draw conclusions you will learn from practice problems in class and in workshops o Connect with the previous module(s) Suggestions:  read the textbook chapter before lecture  go through the lecture pdf (and your notes) after lecture  participate in class  ask questions  Different types of nucleic acids: DNA vs RNA Structure of nucleotides DNA structure Structure of DNA & function Important properties of DNA Some techniques in DNA research Mendel peas and heredity How are inherited traits passed on? (on the molecular level) The search for the heredity molecule What would be some properties of the perfect heredity molecule? The search for the heredity molecule The “transforming principle” Protein or DNA? Griffith, 1928 Avery, McCarty & MacLeod, 1940s Hershey & Chase, 1950s The Race for DNA Structure Double helix? Triple helix? Chargaff’s rules The Race for DNA Structure Double helix? Triple helix? Chargaff’s rules The Race for DNA Structure Double helix? Triple helix? Chargaff’s rules Nucleotides are building blocks of DNA (and RNA) BASE NH2 PHOSPHATE N O 5 N O –O P O CH2 O O– N-glycosidic 4 H H 1 bond H 3 2 H OH H SUGAR Deoxyribonucleotide (Ribonucleotide - OH at C2 position) Nitrogenous bases are derivatives of either purine or pyrimidine Pyrimidine ring Cytosine Uracil (RNA) Thymine (2-oxy-4-amino-pyrimidine) (2-oxy-4-oxy-pyrimidine) (2-oxy-4-oxy-5-methyl pyrimidine) Adenine Guanine Purine ring (6-amino-purine) (2-amino-6-oxy-purine) Bases linked to a sugar (ribose or deoxyribose) form nucleosides N-glycosidic bond syn Guanosine anti Guanosine anti Uridine o Pyrimidines are in anti conformation o Purines favor anti conformation o Tautomers of bases (keto/enol; amino/imino) keto enol Nucleosides linked with a phosphate group(s) at C-5 position of the ribose are called nucleotides BASE NH2 PHOSPHATE N O 5 N O –O P O CH2 O– O 4 H H 1 H 3 2 H OH H SUGAR Deoxyribonucleotide Nucleotides are building blocks of DNA (and RNA) Minor nucleosides in DNA Ex: 5-methylcytosine common in eukaryotes (also found in bacteria) Ex: N6-methyladenosine common in bacteria (found in RNA in eukaryotes) Modification is done after DNA synthesis Epigenetic markers  Mark own DNA so that cells can recognize and degrade foreign DNA (prokaryotes)  Mark which genes should be active (eukaryotes) Nucleotides are building blocks of nucleic acids (DNA and RNA) Also have other functions: ATP provides energy for cellular processes GTP for protein synthesis (translation initiation, elongation) CTP for lipid synthesis UTP for carbohydrate metabolism (UDP-glucose) Cyclic nucleotides are secondary messengers in signaling pathways cAMP, cGMP  Nucleic acids are polynucleotide chains (polymers of nucleotides) o Nucleotides in the chain are linked by 3’-5’ phosphodiester bond o Sugar-phosphate backbone is negatively charged o Nucleic acids are linear (no branching) o Nucleic acids are directional (5’ end is different from 3’ end) o Base sequence is always read from 5’ to 3’ Two types of nucleic acids: DNA and RNA RNA DNA o DNA contains 2-deoxyribose instead of ribose o DNA contains thymine instead of uracil o DNA is a double-stranded helix Lehninger Principles of Biochemistry Chemical differences between DNA and RNA have biological significance o Two hydroxyls next to each other (2’, 3’) make RNA more susceptible to hydrolysis DNA lacks the 2’ OH – more stable o Cytosine can spontaneously deaminate to form uracil Why can this be a problem for DNA? Cytosine Uracil Thymine DNA is a double-stranded helix Major groove Minor groove o Two strands in the helix are antiparallel o Hydrogen bonds between bases stabilize the helix stacking of aromatic bases contributes Double helix can be denatured (‘melted’) and renatured (‘re-annealed’) Double helix can be denatured (‘melted’) and renatured (‘re-annealed’) A, B and Z form of double-stranded helix Major and minor grooves Most proteins interact with DNA via major or minor groove Tertiary Structure of DNA o 10 bp per turn of helix in duplex DNA o DNA sometimes has more or less than 10 bp per turn - a supercoiled state, underwound (-) or overwound (+) o Enzymes called topoisomerases or gyrases can introduce or remove supercoils o Circular DNA can be concatenated Topoisomerase I (Top I) can Topoisomerase II (Top II) can remove supercoils by making resolve concatenates by making single-stranded breaks double-stranded breaks Linear DNA can also be positively or negatively supercoiled. How do you think this can happen? (Considering that it is linear – why doesn’t it just rotate to relieve tension - supercoiling?) Eukaryotic DNA is wrapped around nucleosomes  Human DNA’s total length is ~2 meters  This must be packaged into a nucleus that is about 5 micrometers in diameter  This packing is made possible by wrapping the DNA around protein spools called nucleosomes and then packing these in helical filaments  Euchromatin: a less condensed form of chromatin, consisting of 30-nm fibers and looped domains  Heterochromatin: a highly condensed form of chromatin At which point of the cell cycle is DNA the most compacted? Eukaryotic DNA is wrapped around nucleosomes o Core histones: H2A, H2B, H3, H4 (two of each in a nucleosome) o Highly basic proteins; ~20% K or R (why?) o Covalently modified at many different sites (methylation, acetylation) regulates DNA compaction/accessibility 147 o Histone H1 (linker histone) o Histone variants with special functions (γH2AX, H3.3, H2A.Z..) Nucleosomes are a dynamic structure o Nucleosome remodeling complexes slide histone octamers on DNA dsDNA translocases (can shift the DNA around the nucleosome) remove/exchange histone octamers exchange histone subunits o Histone modifying enzymes (introduce or remove covalent modifications on histone tails) Mapping nucleosomes on the DNA Nucleosomes limit access of some proteins to DNA Accessibility to micrococcal nuclease (MNase digestion) dsDNA endonuclease, not sequence specific cuts linker DNA (cannot access wrapped DNA enough to cut); ~160 bp fragments MNase-seq (positional information based on sequence) How can you determine DNA sequence? Sanger sequencing How can you determine DNA sequence? NGS (massively parallel sequencing)