Structure and Synthesis of DNA
- 1. Chapter Two Synthesis and structure of DNA 1
- 2. Chapter Objectives After the end of this chapter the will able to Define how cells synthesis nucleic acids Mention the components of DNA Explain the structure and functions of nucleic acid Discuss different types of nucleic acids Discuss the role of different enzymes in nucleic acid replication Demonstrate the process of DNA replication Define DNA mutation and repairing mechanisms 2
- 3. Introduction Nucleic acids are biopolymers/ polynucleotides/ composed of nucleotide subunits linked by phosphodiester bonds. There are two types of nucleic acids: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). DNA encodes hereditary information, controls cell division, growth & development. Nucleotide triphosphate serves as substrate precursor for biosynthesis of nucleic acids. Nucleotides are linked by nucleophilic attack of 3’-OH of one nucleotide triphosphate on 5’ phosphorus of another nucleotide 3
- 4. Con’t back bone of alternating phosphate groups either deoxyribose or ribose pentose sugar joined by phosphodiester bond. Heterocyclic bases primarily adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U) are linked by hydrogen bonding. These nitrogenous bases are Purine & pyrimidines. DNA has double intertwining helical strands with deoxyribose sugar A, G, C, and T bases. Whereas RNA is single stranded polynucleotides with ribose sugar, A, G, C, and U heterocyclic bases. The base sequences of each DNA strand are complementary so that base pairs are A═T and G≡C. Base pairing in RNAs occurs between bases on the same folded strand. 4
- 5. DNA What is DNA? Is the genetic molecules carrying all the genetic information within chromosome It is complex molecule that contains all of the information necessary to build and maintain an organism. DNA is a molecule that contains the instructions an organism needs to develop, live and reproduce. 5
- 6. The components of nucleotides The monomeric units for nucleic acids are nucleotides Nucleotides are made up of three structural subunits Sugar: ribose in RNA, -deoxyribose in DNA Heterocyclic base Phosphate 6
- 7. Nucleoside, nucleotides and nucleic acids 7 The chemical linkage between monomer units in nucleic acids is a phosphodiester bond.
- 8. DNA structure and properties DNA is stable form of double helix with 2 distinct sizes of grooves major groove & minor groove running in spiral fashion. Most DNA protein associations are in major grooves. A single strand of nucleotides has no helical structure. Helical shape of DNA depends entirely on the pairing & stacking of the bases in the antiparallel strands DNA handed helix. 8
- 9. DNA structure and properties Primary Structure: the base sequence or nucleotide sequence in polydeoxynucleotide chains It is the order of bases on the polynucleotide sequence; the order of bases specifies the genetic code. 9
- 10. Secondary Structure The 3D conformation of the polynucleotides backbone into double helix of DNA. It the relative spatial position of all the atoms of nucleotide residues 10
- 11. DNA Double Helix & Hydrogen bonding DNA is made of two polynucleotide chains, where the backbone is constituted by sugar-phosphate, and the bases project inside. The two chains have anti- parallel polarity. It means, if one chain has the polarity 5’ 3’, the other has 3’ 5’. The bases in two strands are paired through hydrogen bond (H- bonds) forming base pairs (bp). Adenine forms two hydrogen bonds with Thymine from opposite strand and vice-versa. Similarly, Guanine is bonded with Cytosine with three H-bonds. 11
- 12. Based on the observation of Erwin Chargaff that for a double stranded DNA, the ratios between Adenine and Thymine; and Guanine and Cytosine are constant and equals one. Hydrogen bond:-A chemical bond consisting of a hydrogen atom between two electronegative atoms (e.g., oxygen or nitrogen) with one side be a covalent bond and the other being an ionic bond. 12 DNA Double Helix & Hydrogen bonding
- 13. Tertiary structures Many naturally occurring DNA molecules are circular, with no free 5′ or 3′ end. Due to the polarity of the strands of the DNA double helix, the 5′ end of one strand can only join its own 3′ end to covalently close a circle. The structure of DNA does not only exist as secondary structures such as double helices. but it can fold up on itself to form tertiary structures by supercoiling. Tertiary structures DNA refers the supercoiling the 3D arrangement of all atoms of nucleic acid. 13
- 14. DNA Supercoiling DNA supercoiling refers to the over- or under-winding of a DNA strand, and is an expression of the strain on that strand. Supercoiling is important in a number of biological processes, such as compacting DNA. Additionally, certain enzymes such as topoisomerases are able to change DNA topology to facilitate functions such as DNA replication or transcription. There are two types of DNA Super Coiling. Positive DNA Supercoiling Negative DNA supercoiling 14
- 15. Positive DNA Supercoiling Positive supercoiling is the left-handed, coiling of DNA thus winding occurs in the clockwise direction. This process is also known as the "over winding" of DNA. 15
- 16. Negative DNA supercoiling Negative supercoiling is the right-handed coiling of DNA thus winding occurs in the counterclockwise direction. It is also known as the "underwinding" of DNA. 16
- 17. Importance of DNA Supercoiling DNA supercoiling is important for DNA packaging within all cells. Because the length of DNA can be thousands of times that of a cell, packaging this genetic material into the cell or nucleus (in eukaryotes ) is a difficult feat. Supercoiling of DNA reduces the space and allows for much more DNA to be packaged. In prokaryotes, plectonemic supercoils are predominant, because of the circular chromosome and relatively small amount of genetic material. In eukaryotes, DNA supercoiling exists on many levels of both plectonemic and solenoidal supercoils, with the solenoidal supercoiling proving the most effective in compacting the DNA. Solenoidal supercoiling is achieved with histones to form a 10 nm fiber. This fiber is further coiled into a 30 nm fiber, and further coiled upon itself numerous times more. 17
- 18. Con’t DNA packaging is greatly increased during nuclear division events such as mitosis or meiosis, where DNA must be compacted and segregated to daughter cells. Supercoiling is also required for DNA and RNA synthesis. Because DNA must be unwound for DNA and RNA polymerase action, supercoils will result Quaternary structure (40): Interactions of DNA and proteins small molecules are present to stabilize the structure. 18
- 19. Forms of DNA Three major forms: B-DNA A-DNA Z-DNA 19
- 20. B- DNA It is biologically THE MOST COMMON It is a -helix meaning that it has a Right handed, or clockwise, spiral Ideal B-DNA has 10 base pair per turn Base pair are 0.34 nm apart. So complete rotation of molecule is 3.4 nm. Axis passes through middle of each basepairs. 20
- 21. B-DNA Minor Groove is Narrow, Shallow. Major Groove is Wide, Deep. This structure exists when plenty of water surrounds molecule and there is no unusual base sequence in DNA-Condition that are likely to be present in the cells. B-DNA structure is most stable configuration for a random sequence of nucleotides under physiological condition. 21
- 22. A-DNA Right-handed helix Wider and flatter than B-DNA 11 bp per turn Its bases are tilted away from main axis of molecule Narrow Deep major Groove and Broad, Shallow minor Groove. Observed when less water is present. i.e. Dehydrating condition. 22
- 23. Z-DNA A left-handed helix Seen in Condition of High salt concentration. In this form sugar-phosphate backbones zigzag back and forth, giving rise to the name Z-DNA(for zigzag). 12 base pairs per turn. A deep Minor Groove. No Discernible Major Groove. Part of some active genes form Z-DNA, suggesting that Z- DNA may play a role in regulating gene transcription. 23
- 24. Summery on forms of DNA 24
- 25. DNA Replication Cells need to make a copy of DNA before dividing so each daughter cell has a complete copy of genetic information DNA Replication is the normal process of doubling the DNA content of cells prior to normal cell division. Because the genetic complement of the resultant daughter cells must be the same as the parental cell. DNA replication must possess at very high degree of fidelity. 25
- 26. Components of Replication 1. dNTPs: dATP, dTTP, dGTP, dCTP (deoxyribonucleoside 5’-triphosphates) (sugar-base + 3 phosphates) 2. Ds DNA template 3. Primer- short RNA fragment with a free 3´-OH end 4. Enzyme: DNA-dependent DNA polymerase (DDDP), other enzymes, protein factor 5. Mg 2+ (optimizes DNA polymerase activity) 26
- 27. Enzymes Involved in Replication DNA helicases unwind the double helix, the template strands are stabilized by other proteins Single-stranded DNA binding proteins make the template available RNA primase catalyzes the synthesis of short RNA primers, to which nucleotides are added. DNA polymerase III extends the strand in the 5’-to-3’ direction DNA polymerase I degrades the RNA primer and replaces it with DNA DNA ligase joins the DNA fragments into a continuous daughter strand 27
- 28. Unwind DNA helicase enzyme unwinds part of DNA helix stabilized by single-stranded binding proteins prevents dna molecule from closing! DNA gyrase Enzyme that prevents tangling upstream from the replication fork 28
- 29. RNA Primase Adds small section of RNA (RNA primer) to the 3’ end of template DNA Why must this be done? Primase synthesizes short stretches of RNA nucleotides, providing a 3’-OH group to which DNA polymerase can add DNA nucleotides DNA polymerase 3 (enzyme that builds new DNA strand) can only add nucleotides to existing strands of DNA 29
- 30. DNA Polymerase III Build daughter DNA strand by adding new complementary bases 30
- 31. DNA replication DNA Replication is the process by which the DNA of the ancestral cell is duplicated, prior to cell division. Upon cell division, each of the descendants will get one complete copy of the DNA that is identical to its predecessor Synthesis of both new strands of DNA occurs at the replication fork that moves along the parental molecule The replication fork consists of the zone of DNA where the strands are separated, plus an assemblage of proteins that are responsible for synthesis Sometimes referred to as the replisome 31
- 32. DNA replication 32 • The replication fork is the site of DNA replication and, by definition, includes both the DNA and associated proteins
- 33. DNA replication involves several processes: First, the DNA must be unwound, separating the two strands The single strands then act as templates for synthesis of the new strands, which are complimentary in sequence Bases are added one at a time until two new DNA strands that exactly duplicate the original DNA are produced 33 DNA replication
- 34. Con’t The process is called semi-conservative replication because one strand of each daughter DNA comes from the parent DNA and one strand is new The energy for the synthesis comes from hydrolysis of phosphate groups as the phosphodiester bonds form between the bases 34
- 35. Direction of Replication The enzyme helicase unwinds several sections of parent DNA At each open DNA section, called a replication fork, DNA polymerase catalyzes the formation of 5’-3’ester bonds of the leading strand The lagging strand, which grows in the 3’-5’ direction, is synthesized in short sections called Okazaki fragments The Okazaki fragments are joined by DNA ligase to give a single 3’-5’ DNA strand 35
- 36. Mechanism of DNA Replication DNA replication is catalyzed by DNA polymerase III which needs an RNA primer DNA polymerase III cannot initiate the synthesis of a polynucleotide, they can only add nucleotides to the 3 end The initial nucleotide strand is an RNA primer RNA primase synthesizes primer on DNA strand DNA polymerase adds nucleotides to the 3’ end of the growing strand By: Asmamaw Menelih 36 DNA polymerase I degrades the RNA primer and replaces it with DNA DNA polymerase III adds nucleotides to primer
- 37. DNA Replication By: Asmamaw Menelih 37 DNA polymerase I degrades the RNA primer and replaces it with DNA DNA polymerase III adds nucleotides to primer
- 38. Mechanism of DNA Replication Nucleotides are added by complementary base pairing with the template strand During replication, new nucleotides are added to the free 3’ hydroxyl on the growing strand The nucleotides (deoxyribonucleoside triphosphates) are hydrolyzed as added, releasing energy for DNA synthesis. The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells. 38
- 39. 39 Mechanism of DNA Replication
- 40. The process of DNA replication The process of DNA replication follows the three main steps: 1. Initiation, 2. Elongation, 3. Termination 40
- 41. Initiation Involves recognition of the origin by a complex of proteins. Before DNA synthesis begins, the parental strands must be separated and (transiently) stabilized in the single-stranded state. Then synthesis of daughter strands can be initiated at the replication fork by RNA primer. 41
- 42. Elongation Is undertaken by another complex of proteins. Involves the addition of new nucleotides (dNTPs ) based on complementarity of the template strand Forms phosphoester bonds, Correct the mismatch bases, extending the DNA strand 42 DNA polymerase III
- 43. Termination At the end of the replicon, joining and/or termination reactions are necessary. Following termination, the duplicate chromosomes must be separated from one another, which requires manipulation of higher-order DNA structure. The terminal structure of eukaryotic DNA of chromosomes is called telomere. Telomere is composed of terminal DNA Sequence and protein The sequence of typical telomeres is rich in T and G The telomere structure is crucial to keep the termini of chromosomes in the cell from becoming entangled and sticking to each other. 43
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- 45. Replication in prokaryotic Vs. eukaryotic No. DAN replication in prokaryotic DNA replication in eukaryotic 1 It occurs inside the cytoplasm It occurs inside the nucleus 2 Have Only one origin of replication per DNA molecule Many origin of replication (over 1000) in each chromosome 3 Origin of replication is formed of about 100-200 or more nucleotides Each origin of replication is formed of about 150 nucleotides 4 Replication of DNA occurs at one point in each DNA molecule Occurs at several points simultaneously in each chromosome 5 Prokaryotic chromosome has one replicon Eukaryotic DNA molecules have large number of replicons(50000 and above), but replication does not occur simultaneously on all replicons 6 One replication bubble is formed during DNA replication Numerous replication bubbles are formed in one replicating DNA molecule 7 Initiation of DNA replication is carried out by protein DnaA and DnaB Initiation is carried out by multi-sub-unit protein, origin recognition complex 45
- 46. Cont.. 8 DNA gyrase is needed DNA gryase is needed 9 Okazaki fragment are large, 1000-2000 nucleotides long Okazaki fragment are short, 100-200 nucleotides long 10 Replication is very rapid, some 2000 bp second are added Replication is slow, some 100 nucleotides per second are added 46 11
- 47. DNA mutation and repair What is a mutation? Changes in the nucleotide sequence of DNA May occur in somatic cells (aren’t passed to offspring) May occur in gametes (eggs & sperm) and be passed to offspring 47
- 48. Are Mutations Helpful or Harmful? Mutations happen regularly Almost all mutations are neutral Chemicals & UV radiation cause mutations Many mutations are repaired by enzymes Some type of skin cancers and leukemia result from somatic mutations Some mutations may improve an organism’s survival (beneficial) 48
- 49. What is a mutation? Substitution, deletion, or insertion of a base pair. Chromosomal deletion, insertion, or rearrangement. Somatic mutations occur in somatic cells and only affect the individual in which the mutation arises. Germ-line mutations alter gametes and passed to the next generation. Mutations are quantified in two ways: 1. Mutation rate = probability of a particular type of mutation per unit time (or generation). 2. Mutation frequency = number of times a particular mutation occurs in a population of cells or individuals. 49
- 50. Type of Mutations Transition: One purine replaced by a different purine;or one pyrimidine replaced by a diferent pyrimidine A G T C Transversion: A purine replaced by a pyrimidine or vice versa I. Point mutation: A. Base substitution A T Change in DNA C G 50
- 51. Type of Mutations … B. Change in protein 1. Silent mutation: altered codon codes for the same a.a. 2. Neutral mutation: altered codon codes for functional similar a.a. 3. Missense mutation: altered codon codes for different dissimilar a.a. 4. Nonsense mutation: altered codon becomes a stop codon GAG (Glu) --->GAA (Glu) GAG--->GAC (Asp) or (DE) GAG ---> AAG (Lys) GAG ---> UAG (stop) 51
- 52. Type of Mutations … Frameshift mutation: addition or deletion of one base-pair result in a shift of reading frame and alter amino acid sequence 52
- 53. Types of chromosomal mutations Inversion 53
- 54. Replication Fidelity Replication based on the principle of base pairing is crucial to the high accuracy of genetic information transfer. Enzymes use two mechanisms to ensure the replication fidelity Proofreading and real time correction Base selection 54
- 55. DNA repair mechanisms Enzyme-based repair mechanisms prevent and repair mutations and damage to DNA in prokaryotes and eukaryotes. Types of mechanisms DNA polymerase proofreading - 3’-5’ exonuclease activity corrects errors during the process of replication. Photoreactivation (also called light repair) - photolyase enzyme is activated by UV light (320-370 nm) and splits abnormal base dimers apart. 55
- 56. Con’t Demethylating DNA repair enzymes - repair DNAs damaged by alkylation. Nucleotide excision repair (NER) - Damaged regions of DNA unwind and are removed by specialized proteins; new DNA is synthesized by DNA polymerase. Methyl-directed mismatch repair - removes mismatched base regions not corrected by DNA polymerase proofreading. Sites targeted for repair are indicated in E. coli by the addition of a methyl (CH3) group at a GATC sequence. 56
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