Nucleic Acids I: DNA PDF

Nucleic Acids I: DNA [1] NUCLEIC ACIDS I: DNA Paul J. McDermott, Ph.D. Office: 843 792-3462 Email: [email protected] A. MOLECULAR STRUCTURE OF DNA 1. Polynucleotide Chain 2. Polarity of DNA Strands 3. Flexibility of DNA Strands 4. Shorthand Notation for DNA Strands B. DNA DOUBLE HELIX 1. Chargaff’...

Nucleic Acids I: DNA [1] NUCLEIC ACIDS I: DNA Paul J. McDermott, Ph.D. Office: 843 792-3462 Email: [email protected] A. MOLECULAR STRUCTURE OF DNA 1. Polynucleotide Chain 2. Polarity of DNA Strands 3. Flexibility of DNA Strands 4. Shorthand Notation for DNA Strands B. DNA DOUBLE HELIX 1. Chargaff’s Rule 2. X-ray Diffraction of DNA 3. Watson & Crick Model 4. Complementary Base Pairing C. STRUCTURAL FORMS OF DNA 1. A-DNA 2. B-DNA 3. Z-DNA D. DNA SUPERCOILING 1. Linking Number (Lk) 2. Supercoiling of DNA in Chromosomes 3. Topoisomerases 4. Functional Significance of Supercoiling 5. Targeting Topoisomerases in Medicine E. PHYSICAL PROPERTIES OF DNA 1. Forces Contributing to the Stability of the Double Helix 2. Absorbance of UV Light 3. DNA Melting Temperature 4. Denaturation and Reannealing 5. Binding to Complementary DNA Sequences by Hybridization Suggested Reading: Marks' Basic Medical Biochemistry, 5th Ed: Chapter 12, P. 216-221 “We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.). This structure has novel features which are of considerable biological interest.” James Watson and Francis Crick Nucleic Acids I: DNA [2] OBJECTIVES 1. Specify the following features of the B form of DNA: 3,5´phosphodiester bonds of the sugar-phosphate backbone, number of hydrogen bonds in complementary base pairings, major and minor grooves, handedness. 2. Describe the following physical dimensions of the DNA double helix (B form): diameter, number of bases per turn, approximate distance between stacked bases. 3. Explain what is meant by polarity of complementary DNA strands. 4. Predict the complementary strand of DNA using the same shorthand notation. Example: pCpGpGpTpApApT 5. State Chargaff's rule and explain how it can be used to infer the base composition of a given molecule of DNA. 6. Define relaxed and supercoiled DNA. Describe the relationship between Linking Number (Lk) and positive versus negative supercoiling. 7. Specify the effects of Type I and Type II Topoisomerases on Linking Number (Lk). 8. Describe the functional significance of DNA supercoiling and the role of Type I and Type II Topoisomerases. 9. Specify the 3 main forces that contribute to stabilization of the DNA double helix. 10. Define melting temperature (Tm) and how it can be used to predict G+C content of DNA. 11. Describe how the properties of denaturation and reannealing are used for detection of specific DNA sequences. Illustrations adapted from: • Marks’ Basic Medical Biochemistry: A Clinical Approach © 2012 Lippincott Williams & Wilkins • Biochemistry © 2002 by W.H. Freeman and Company • Lehninger Principles of Biochemistry © 2005 by W.H. Freeman and Company Nucleic Acids I: DNA [3] A. MOLECULAR STRUCTURE OF DNA 1. Polynucleotide Chain Each DNA strand is a chain of purine and pyrimidine deoxyribonucleotides linked by 3´ to 5´ phosphodiester bonds. 2. Polarity of DNA Strands 5´-PO4 3´-OH Each DNA strand has a 5´-end with a phosphate group and a 3´-end with an OH group. N-Glycosidic bond 3. Flexibility of DNA Strands The sugar-phosphate backbone of DNA strands is flexible due to rotation of 3´ to 5´ phosphodiester bonds. 4. Shorthand Notation for DNA Strands 5´-A-C-G-T-3´ or pApCpGpT p indicates 5´-end of the strand B. DNA DOUBLE HELIX 1. Chargaff’s Rule • Content of A = Content of T • Content of G = Content of C 2. X-ray Diffraction of DNA: Rosalind Franklin Stack of nucleotide bases - 3.4 Å spacing Helix Using x-ray diffraction, Rosalind Franklin discovered that DNA can exist in 2 forms: the dehydrated A form and hydrated B form. She used the B form to generate the classic image labeled “photo 51” shown here. She noted that the B form appears to have a helical structure, but still decided to study the A form first. Nucleic Acids I: DNA [4] B. DNA DOUBLE HELIX 3. Watson & Crick Model 5´ 3´ 5´ • Double helix is formed by 2 antiparallel strands of DNA that run in opposite directions 3´ • DNA strands consist of a deoxyribose-phosphate backbone linked by 3´ to 5´ phosphodiester bonds. • 2 strands of double helix are complementary due to hydrogen bonding between A-T and C-G base pairs in the interior the molecule. • Double helix is right-handed around the central axis. The diameter of helix is 20 Å. • Complementary DNA strands in the double helix remain equidistant throughout entire length of the molecule and are twisted to form major and minor grooves. Major Groove Minor Groove • Base pairs extend in a plane perpendicular to the axis of the helix. • Bases are stacked approximately 3.4 Å apart. • 10.4 base pairs per turn of helix • 36 Å per turn of helix (3.4 Å/base x 10.4 bases/turn) 5´ 3´ 5´ 3´ 4. Complementary Base Pairing • G-C base pairs formed by 3 H+ bonds; A-T base pairs formed by 2 H+ bonds. • The glycosidic bonds of both strands in the double helix are rotated to the anti conformation. • Major and minor grooves occur in DNA because the glycosidic bonds are not diametrically opposed to each other in the double helix. Adenine-Thymine Guanine-Cytosine Nucleic Acids I: DNA [5] C. STRUCTURAL FORMS OF DNA 1. A-DNA Dehydrated DNA and DNA-RNA hybrids 2. B-DNA Hydrated DNA (Watson & Crick model) 3. Z-DNA Regions of alternating Pyr and Pur [CGCGCG] • Right-handed double helix • Right-handed double helix • Left-handed double helix • 2.6 Å base stacking • 3.4 Å base stacking • 3.8 Å base stacking • 11 bases per turn • 10.4 bases per turn • 12 bases per turn D. DNA SUPERCOILING DNA molecules are long polymers that are subjected to torsional stress caused by either underwinding or overwinding of the strands around the central axis of the double helix. 1. Linking Number (Lk ): Number of times a DNA strand winds around the helical axis • Twisting of DNA segment to cause underwinding of the strands decreases Lk. • Twisting of DNA segment to cause overwinding of the strands increases Lk. helical axis Relaxed: Lk = 4 Underwinding Lk = 2 Overwinding Lk = 8 - supercoil + supercoils • Torsional stress is relieved by bending the helical axis of a DNA segment to generate supercoils. • Overwinding of DNA strands is relieved by positive supercoiling, while underwinding of DNA strands is relieved by negative supercoiling. • Positive and negative supercoils are generated as a result of strand separation that occurs during processes such as DNA replication, transcription and homologous recombination. Nucleic Acids I: DNA [6] D. DNA SUPERCOILING 2. Supercoiling of DNA in Chromosomes In order for processes such as DNA replication and transcription to occur, the DNA strands must be separated at specific sites in a chromosome by unwinding of the double helix. If chromosomal DNA had free ends, unwinding would not generate torsional stress because the strands could freely rotate about each other as they separate. However, chromosomal DNA does not have free ends because it is either circular (prokaryotes) or consists of long, linear molecules (eukaryotes) that are packaged into higher order chromatin structure and attached to a protein scaffold. a) Circular Chromosome (prokaryotes) helical axis helical axis Unwind DNA by 1 turn around helical axis to reduce the Linking Number (Lk) Torsional stress caused by unwinding of DNA is relieved by bending of helical axis to produce a negative supercoil b) Linear Chromosomes (eukaryotes) • The figure in the lower left shows the effect of unwinding DNA strands that have free ends. In this example, unwinding the DNA strands by 1 turn around the helical axis reduces Lk by 1. Torsional stress would not occur because the free ends of the DNA strands can rotate freely around each other. Thus, a supercoil would not be generated. • The figure in the lower right shows the effect of unwinding DNA strands between 2 fixed ends, for example, during transcription. Unwinding DNA at one site in the chromosome by 1 turn would result in overwinding (increase in Lk) in the region of DNA immediately ahead of it as the strands are fixed and cannot rotate around each other. The increase in Lk creates torsional stress that would be relieved by bending the helical axis to generate a positive supercoil. Fixed End Free End Unwind DNA by 1 turn to separate strands Fixed End Fixed End Unwind DNA by 1 turn to separate strands 3. Topoisomerases Both prokaryotes and eukaryotes have enzymes called topoisomerases that detect torsional stress associated with supercoiling and change the Lk. These enzymes cleave DNA strands to create free ends, thereby allowing the strands to rotate around the helical axis. Topoisomerases are essential for "relaxing" positive and negative supercoils that can be generated during 1. Detect torsional stress processes such as DNA replication, recombination and transcription. 2. Cleave DNA strands 3. Relax +ve/-ve supercoils Nucleic Acids I: DNA [7] D. DNA SUPERCOILING 3. Topoisomerases a) Type I Topoisomerases: Enzymes that change Lk by cleaving a single DNA strand, passing the other intact DNA strand through the gap and resealing the DNA strand. Some Type I Topoisomerases operate as “swivelases” by cleaving a single DNA strand, rotating the free end around the intact DNA strand and resealing the DNA strand. b) Type II Topoisomerases: Enzymes that are ATP-dependent and change Lk by cleaving both DNA strands, passing intact DNA through the gap, and resealing both DNA strands. Prokaryotes have a special Type II Topoisomerase called Gyrase that can create negative supercoils by reducing the Lk. The ability to create negative supercoiling is essential in bacteria for compacting the circular chromosome. Topoisomerase Prokaryotes Eukaryotes Type I Relax negative supercoils (↑ Lk) Relax negative supercoils (↑ Lk) Relax positive supercoils (↓ Lk) Type II Relax positive supercoils (↓ Lk) Relax negative supercoils (↑ Lk) Relax positive supercoils (↓ Lk) Gyrase Type II Topoisomerase that creates negative supercoils (↓ Lk) None present 4. Functional Significance of DNA Supercoiling a) Genomic DNA: In general, DNA is negatively supercoiled in cells by packaging into chromatin (refer to notes on Organization of the Genome). The wrapping of DNA into solenoids results in underwinding (reduced Lk) that is stabilized by histones and other chromatin proteins. Negative supercoiling provides several functional advantages for genomic DNA: • Compaction of long, linear DNA molecules with reduced torsional stress • Easier to unwind negatively supercoiled DNA than positively supercoiled DNA • Stored energy to facilitate unwinding of DNA for replication, transcription and other processes. b) Transcription: RNA Polymerases unwind DNA to form a "transcription bubble" (refer to notes on transcription). Ahead of the bubble, overwinding occurs that can cause positive supercoils. Behind the transcription bubble, negative supercoils can form as the DNA rewinds. c) DNA Replication: DNA Polymerases unwind DNA to form replication forks consisting of 2 template strands (refer to notes on DNA replication). Overwinding ahead of the fork can cause formation of positive supercoils; newly replicated DNA behind the fork can form positive or negative supercoils that can lead to formation of interlinked loops (catenanes). d) Mitosis and Meiosis: DNA is highly compacted in concert with proteins during prophase in order to form metaphase chromosomes. This process can generate positive supercoils that must be relaxed for proper chromosome segregation. Nucleic Acids I: DNA [8] D. DNA SUPERCOILING 5. Targeting Topoisomerases in Medicine a) Antibiotics: Bacterial gyrase is a target of antibiotics such as nalidixic acid and ciprofloxacin. Gyrase inhibitors are used to treat urinary tract infections and other infections such as Bacillus anthracis (anthrax). b) Chemotherapy: Rapidly dividing cells require topoisomerase activity for DNA replication. • Type I Topoisomerase Inhibitors: Topotecan and Irinotecan are derivatives of a naturally occurring compound called Camptothecin. • Type II Topoisomerase inhibitors: Etoposide and Doxorubicin Hydrophobic interior E. PHYSICAL PROPERTIES OF DNA 1. Forces Contributing to Stability of the Double Helix 5´ a) Hydrophobic interior & Hydrophilic exterior Stacked bases inside the double helix are hydrophobic and completely surrounded by the sugar-phosphate backbone, which is hydrophilic due to the electronegative oxygen molecules of phosphodiester bonds. 3´ b) Complementary Base Pairing 2 DNA strands of the double helix are held together equidistantly by hydrogen bonds between G-C and A-T. Hydrophilic exterior c) Base Stacking interactions Double helix is stabilized vertically by the additive effects of van der Waals forces between stacked bases. 2. Absorbance of UV Light 3´ 5´ Hyperchromism: Increased absorbance of UV light due to unstacking of base pairs Nucleic Acids I: DNA [9] E. PHYSICAL PROPERTIES OF DNA 3. DNA Melting Temperature Tm = temperature at midpoint of the transition Tm is determined by G+C content of DNA 4. Denaturation and Reannealing a) Denaturation: Unwinding strands of DNA double helix • Heating • Extremes in pH • Denaturing reagents, e.g., urea A-T rich regions denature first Heat b) Reannealing: Strands hybridize by complementary base pairing Cooperative unwinding of DNA strands Cool Strand Separation 5. Binding to Complementary DNA Sequences by Hybridization • The properties of denaturation and reannealing to complementary sequences are used routinely in research and clinical medicine for several techniques, e.g., sequence-specific primers in PCR, priming of DNA sequencing reactions, detection of sequences in fragments on Southern blots, and detection of sequences in chromosomes or cells by in situ hybridization. • In the example below, DNA is denatured to separate the strands and hybridized to a probe containing a complementary DNA sequence. The probe would be radiolabeled or tagged with a fluorescent molecule for detection of the DNA sequence. 5´3´- -3´ -5´ Denaturation 5´- -3´ & DNA Strands 3´- -5´ Hybridization to complementary probe 3´- 5´-C-G-A-T-C-G-A-T-C-G-A-T-C-G-A-T-C-G-A-T -3´ G-C-T-A-G-C-T-A-G-C-T-A-G-C-T-A-G-C-T-A -5´ DNA Strand