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1. CELL CYCLE

2. CELL CYCLE A cell cycle is a series of events that a cell passes through from the time until it reproduces its replica. It is the growth and division of single cell into two identical daughter cells. In prokaryotic cells, the cell cycle occurs via a process termed binary fission. In eukaryotic cells, the cell cycle can be divided in two periods: INTERPHASE and MITOSIS. The duration of cell cycle varies from hours to years. A typical human cell has a duration of 24 hours. Fig: Over view of Cell cycle

3. PHASES OF CELL CYCE It consists of 2 major activities. G 1 (pre-synthetic phase) S (DNA synthesis) G 2 (pre-mitotic phase) INTER PHASE It includes division of the cell nucleus (mitosis) and division of the cell cytoplasm (cytokinesis) MITOSIS (M PHASE)

4. INTERPHASE It is the longest phase. In a typical human cell out of 24hrs, interphase last for 23hrs. It is also known as resting phase of the cell cycle. During this phase mRNA and rRNA are synthesized. The chromosomes duplicates into two chromatids . The centrospheres of centrioles and microtubules arise. Stage of interphase Interphase consists of 3 sub-stages G1 PHASE S PHASE G2 PHASE

5. also called as cell differentiation In this phase cell quit from cell cycle hence also called as phase of cell quiescence In these cells cyclin D is in decreased concentration . Rb protein is in hypo-phosphorylated (active form). GO PHASE G1 PHASE ( first gap ) It is also called the growth phase.(longest phase) During this phase 20 amino are formed, from which millions of proteins and enzymes are formed, which are required in S phase. Cell is preparing for S phase During this phase mRNA, rRNA and tRNAs are formed. Concentration of cyclin D increases. Formation of cyclin E complex is necessary.

6. During this phase DNA synthesis occurs. histone synthesis Cyclin E/CDK and cyclin A/CDK regulate the processes in phase S. Cyclins, when bound with the dependent kinases such asCdk1 proteins form the Maturation- Promoting Factor ( MPF ). S PHASE (DNA synthesis) It is the second growth phase. Cells continue to grow and produce new proteins. The nucleus increases in volume. mRNA , tRNA and rRNA synthesis also occur. This phase has double the number of chromosomes. Cyclin A/ cdk and cyclin B/ cdk complexes are active which are necessary for the cell to enter into M phase (check point 2). G2 PHASE (pre-mitotic phase)

7. M PHASE (division phase) KARYOKINESIS CYTOKINESIS

8. CELL CYCLE CHECKPOINTS (RESTRICTION POINTS) 1.G 1 checkpoint (G 1 restriction point) 2.G 2 checkpoint. 3.M checkpoint. A checkpoint is a stage in the eukaryotic cell cycle at which the cell examines internal and external cues and " decides " whether or not to move forward with division.

9. G 1 checkpoint (G 1 restriction point) It prevents DNA damage from being replicated. This restriction point is mainly controlled by the action of the CDKI-p16 ( CDK inhibitor p16 ). The inhibited CDK not bind with cyclin D1 , hence there is no cell progression. Active cyclin D- cdk complexes phosphorylate retinoblastoma protein ( pRb ) in the nucleus. Once pRb gets phosphorylated, E2F activates the transcription of cyclins E and A, which then interacts with CDK2 to allow for G 1 -S phase transition. G1 checkpoint Checks for ; Cell size Nutrients Growth factor DNA damage

10. G 2 checkpoint This checks the number of factors which are essential for the cell division. MPF ( Maturation-promoting factor ) promotes the G 2 phase into the entrance of M-phase. The main functions of MPF in this restriction point are : Triggers the formation of mitotic spindle. Promotes chromosome condensation. Causes nuclear envelop breakdown. If there are any damages are noticed in this restriction point, then the phosphatase not activate the MPF, resulting in the arrest of cell cycle in G 2 phase till the repair of the damaged DNA.

11. M checkpoint It is also known as spindle checkpoint. This occurs at metaphase. Anaphase-promoting complex ( APC ) regulates this checkpoint. Check whether the sister chromatids are correctly attached to the spindle microtubules If there are mistakes then it delays the cell in entering into anaphase from metaphase.

12. What happens if you lose one of checkpoints? DNA replication  and  chromosome distribution  are indispensable events in the cell cycle control. Cells must accurately copy their chromosomes, and through the process of mitosis, segregate them to daughter cells. The checkpoints are  surveillance mechanism  and  quality control  of the genome to maintain genomic integrity. Checkpoint failure often causes mutations and genomic arrangements resulting in genetic instability .  Checkpoint studies are very important for understanding mechanisms of  genome maintenance  as they have direct impact on  the ontogeny of birth defects  and the  cancer biology.

13. CYCLINS . The cyclin in the mammalian are roughly divided according to their activity in the different phase of the cell cycle. The G1/S cyclin includes the D and E type cyclin. The M phase specific cyclin includes the B type cyclins. Cyclin of type A are active in S, G2 and M phase CELL CYCLE REGULATORS cyclin D cyclin A cyclin E cyclin B Cyclin are basically some protein which helps in cell cycle regulation with their partner CDKs . Cyclin of type D, A, E and B shows characteristic concentration change in the course of cell cycle Function of cyclin: Activation of CDK. contribution to substrate specificity of CDKs. Regulation of cyclin expression .

14. The attached phosphate group acts like a switch, making the target protein more or less active. When a cyclin attaches to a Cdk , it has two important effects: it activates the Cdk as a kinase, but it also directs the Cdk to a specific set of target proteins, G1/S cyclins send Cdks to S phase targets ( e.g., promoting DNA replication ) M cyclins send Cdks to M phase targets (e.g., making the nuclear membrane break down ). How does this work? PHASE CYCLIN CDK GO C CDK3 GI D,E CDK4, CDK2, CDK6 S A,E CDK2 G2 A CDK2, CDK1 M B CDK I CDKs ( Cyclin Dependent Kinases) CDKs are kinases, enzymes that phosphorylate ( attach phosphate groups to ) specific target proteins.

15. Cancer and the cell cycle Cancer cells may make their own growth factors Cancer cells can divide many more times than this, largely because they express an enzyme called  telomerase . cancer cells gain the ability to migrate to other parts of the body, a process called   metastasis , and to promote growth of new blood vessels, a process called  angiogenesis  ( which gives tumor cells a source of oxygen and nutrients ).   Cancer is basically a disease of uncontrolled cell division. Its development and progression are usually linked to a series of changes in the activity of cell cycle regulators . How cancer develops? Most cancers arise as cells acquire a series of mutations If a cell lose the activity of a cell cycle inhibitor = form BENIGN TUMOR Increased activity of a positive cell cycle regulator in one of the descendants cells may give rise to MALIGNANT TUMOR

16. Cell cycle regulators and cancer Mutations of two types of cell cycle regulators may promote the development of cancer: positive regulators may be overactivated (become oncogenic), while negative regulators, also called tumor suppressors, may be inactivated. ONCOGENES The overactive ( cancer-promoting ) forms of these genes are called oncogenes, while the normal, not-yet-mutated forms are called proto-oncogenes.  Mutations that turn proto-oncogenes into oncogenes can take different forms .  change the amino acid sequence of the protein. Amplification or an error in DNA repair. the growth factor receptor, the Ras protein, and the signalling enzyme Raf are all encoded by proto-oncogenes. Overactive forms of these proteins are often found in cancer cells. oncogenic Ras mutations are found in about 90% of pancreatic cancers.  Cancer-causing mutations often change Ras’s structure so that it can no longer switch to its inactive form, or can do so only very slowly, leaving the protein stuck in the “on” state.

17. TUMOR SUPPRESSORS Genes that normally block cell cycle progression are known as  tumor suppressors. Tumor suppressors prevent the formation of cancerous tumors when they are working correctly, and tumors may form when they mutate so they no longer work. One of the most important tumor suppressors is  tumor protein p53 When a cell’s DNA is damaged, a sensor protein activates p53, which halts the cell cycle at the G1, checkpoint by triggering production of a  cell-cycle inhibitor activate DNA repair enzymes triggering apoptosis (programmed cell death) so that damaged DNA is not passed on. In cancer cells, p53 is often missing, non functional, or less active than normal.   For example, many cancerous tumor have a mutant form of p53 that can no longer bind DNA. When p53 is defective, a cell with damaged DNA may proceed with cell division.

18. APOPTOSIS VS. NECROSIS usually “spill their guts” as they die. the cell swells up. Causes inflammation in the tissue. They shrink and develop bubble-like protrusions (“blebs”) on their surface. the DNA in the nucleus gets chopped up into small pieces, and some organelles of the cell, In the end, the entire cell splits up into small chunks . Necrosis (the messy way) Apoptosis  is a form of programmed cell death , or “ cellular suicide .” Greek word “ apo ” meaning “ away ” and “ ptosis ” meaning “ falling ”. APOPTOSIS Apoptosis (the tidy way)

19. In order to remove cells that should no longer be part of the organism. Some cells need to be “deleted” during development. Abnormal cells Cells in an adult organism may be eliminated to maintain balance. Apoptosis is part of development: Why do cells undergo apoptosis? in the worm  C. Elegans , 131 cells will die by apoptosis as the worm develops from a single cell to an adult. Apoptosis also plays a key role in human development. Other examples of apoptosis during normal development include the loss of a tadpole’s tail as it turns into a frog.

20. Apoptosis is key to immune function: development and maintenance of a healthy immune system. Apoptosis also plays an important role in allowing the immune system to turn off its response to a pathogen. Apoptosis can eliminate infected or cancerous cells: When a cell’s DNA is damaged, it will typically detect the damage and try to repair it.

21. REFERENCE: A Textbook Of “ Cell And Molecular Biology ” Author SP Vyas , A Mehta First Edition 2011 , CBS Publishers & Distributors Pvt. Ltd . A textbook of “Genetics” Author P.S VERMA ,V.K AGARWAL 9 th Revised Multicolour First Edition 1975. P rinted Rajendra Ravindra printers Pvt.Ltd; Published by S CHAND & COMPANY Pvt.Ltd A text book of “ cell biology ” Author T DEVASENA First edition April 2012 publisher; OXFORD UNIVERSITY PRESS. ‘ ’Cell Biology, Genetics, Molecular Biology, Evolution & Ecology ’’ Author   P.S VERMA . ,  V.K. AGARWAL Publisher:   S Chand; Reprint Edition. 2006 edition (1 September 2004)

22. Synthesis and structure of DNA 1

23. 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

24. 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

25. 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

26. DNA 5

27. 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

28. Nucleoside, nucleotides and nucleic acids 7 The chemical linkage between monomer units in nucleic acids is a phosphodiester bond .

29. 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

30. DNA structure and properties Structure: sequence the base sequence or in polydeoxynucleotide ▶ Primary nucleotide chains ▶ It is the order of bases on the polynucleotide sequence; the order of bases specifies the genetic code. 9

31. 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

32. 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

33. 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

34. 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

35. 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

36. 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

37. 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

38. 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

39. 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 (4 ): Interactions of DNA and proteins small molecules are present to stabilize the structure. 18

40. Forms of DNA Three major forms: B- DNA A-DNA Z- DNA 19

41. 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

42. 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

43. 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

44. 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

45. Summery on forms of DNA 24

46. 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

47. Components of Replication dNTPs : dATP, dTTP, dGTP, dCTP (deoxyribonucleoside 5 ’ -triphosphates) (sugar- base + 3 phosphates) Ds DNA template Primer - short RNA fragment with a free 3´-OH end Enzyme : DNA- dependent DNA polymerase (DDDP), other enzymes, protein factor Mg 2+ (optimizes DNA polymerase activity) 26

48. 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

49. 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

50. 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

51. DNA Polymerase III 30 Build daughter DNA strand by adding new complementary bases

52. 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

53. DNA replication 32 The replication fork is the site of DNA replication and, by definition, includes both the DNA and associated proteins

54. ▶ 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

55. 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

56. 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

57. 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

58. 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

59. 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

60. 39 Mechanism of DNA Replication

61. The process of DNA replication The process of DNA replication follows the three main steps: Initiation, Elongation, Termination 40

62. 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

63. 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

64. 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 from becoming The telomere structure is crucial to keep the termini of chromosomes in the cell entangled and sticking to each other. 43

65. 44

66. 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

67. Cont.. 46 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 11

68. 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

69. 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

70. 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 : Mutation rate = probability of a particular type of mutation per unit time (or generation). Mutation frequency = number of times a particular mutation occurs in a population of cells or individuals. 49

71. Type of Mutations I. Point mutation: A. Base substitution Change in DNA 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 A T C G 50

72. 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 (D  E) GAG --- > AAG (Lys) GAG --- > UAG (stop) 51

73. 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

74. Types of chromosomal mutations Inversion 53

75. 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

76. 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

77. 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 (CH 3 ) group at a GATC sequence. 56

78. TRANSCRIPTION & TRANSLATION

79. DEFINITION :- Transcription is synthesis of single stranded RNA from a double stranded DNA template . Its produces messenger RNA ( mRNA) . Translation is the 1 st stage of protein biosynthesis from RNA . In this process formation of a polypeptide by using mRNA as a template. It occurs in ribosomes . Transcription and Translation both process are the part of gene expression.

80. Cont… In a eukaryotic cell the nuclear envelope separates transcription from translation. Extensive RNA processing occurs in the nucleus.

81. TRANSCRIPTION INTRODUCTION :- Transcription is the synthesis of mRNA from a DNA template which occurs in 5’-3’ direction. During transcription , a DNA sequence is read by an RNA polymerase which produce a complementary and antiparallel RNA strand. Transcription is the first step leading to gene expression.

82. Cont… The stretch of DNA transcribed into an RNA molecule is called as transcription unit which encoded at least one gene. The result of the transcription is a mRNA which will then be used to create that protein via the process of translation

83. Transcription Prokaryotes Vs Eukaryotes. Prokaryotic transcription occurring in cytoplasm alongside translation and eukaryotic transcription occurring only in the nucleus where it is separated from the cytoplasm by the nuclear membrane. Eukaryotic DNA not currently used in stored as heterochromatin around histones to from nucleosomes and must be unwound as euchromatin to be transcribed.

84. STAGES OF TRANSCRIPTION :- There are three stages involved in transcription :- INITIATION ELONGATION TERMINATION

85. INITIATION:- RNA polymerase binds to specific DNA region and initiate transcription called as promoter site . RNA polymerase is the enzyme responsible for transcription. It have 5 subunits:- 2 α subunit, β subunit, β ’subunit, ω subunit. After polymerase is bound to the promoter DNA, the two DNA strands unwind and the enzyme starts transcribing the template strand.

86. Cont… The position of the first synthesized base of the RNA is called the start site .

87. ELONGATION:- RNA polymerase moves along DNA template and sequentially synthesizes the RNA chain. DNA is unwinding ahead of the moving polymerase and the helix is reformed behind it. It unwinds 10-20 DNA bases at a time. RNA polymerase add nucleotides in the 5’-3’ direction. The new section of RNA ‘peels away’ as the double helix reforms.

88. TERMINATION:- Transcription stops when RNA polymerase reaches a section of DNA called the terminator. Terminator sequence = AAUAAA. Next, the RNA strand is released and RNA polymerase dissociates from the DNA. The RNA strand will go through more processing.

89. RNA Processing :- The orignal transcript from the DNA is called as pre-m RNA. It contains transcript of both intron and exons . Intron:- it is non-coding sections of nucleic acid found between coding regions. Exons: - coding regions of nucleic acids Pre-mRNA never leaves the cell’s nucleus.

90. Cont… The introns are excised and exons are joined together to form mRNA. The introns are removed by a process called splicing to produce messenger RNA (mRNA)

91. TRANSLATION Translation is a process in which the formation of polypeptide (PROTEIN) by decoding mRNA produced in transcription. It occurs in ribosome which are present in cytoplasm. It begins after mRNA enters in cytoplasm. It uses tRNA as the interpreter of mRNA.

92. PHASES IN TRANSLATION :- Translation proceed in four phases:- INITIATION ELONGATION TRANSLOCATION TERMINATION

93. INITIATION :- The initiation stage of translation brings together mRNA, tRNA bearing the first amino acid of the polypeptide, and two subunits of a ribosome The components involved are the large and small subunits of ribosome, mRNA, initiator tRNA in its charged form and three factors ( IF1.IF2,IF3) and GTP. The tRNA has a amino acid linked to it is term as Charged tRNA .

94. Cont… IF1 and IF2 bind to free 30S subunit. IF3 complexed with GTP then bind to the small subunit. It will assist the charged initiator tRNA to bind. The assembled ribosome has 2 tRNA binding sites. These are called A-site (acceptor) for aminoacyl and P-site(donor)for polypeptide. The A-site is where incoming aminoacyl-tRNA moleules bind and P-site where the growing polypeptide chain usually found.

95. Cont… One major outcome of initiation is the placement of initiator tRNA in the P-site. Start codon :- AUG Start anticodon :- UAC The small ribosomal subunit attaches to 5’ end of mRNA.

96. ELONGATION:- In this amino acid are added one by one to the first amino acid called as amino acid delivery. In codon recognition , mRNA codon in the A site forms hydrogen bond with the tRNA anticodon . In peptide bond formation, the ribosome catalyzes the formation of the peptide bond between the amino acids. The polypeptide extending from the P-site moves to A-site to attach to the new Amino Acid. In elongation process three elongation factors (EF-T4,EF-T5,EF-G) which will bind with GTP or GDP.

97. TRANSLOCATION :- The t-RNA with the polypeptide chain in the A site is translocated to the P site. tRNA at the P site moves to the E site and leaves the ribosome. The ribosome moves down the mRNA in the 5’-3’ direction.

98. TERMINATION:- Protein factors called release factors interact with the stop codons and cause release of the completed polypeptide chain. Stop codon – UAA,UAG,UGA. RF1 recognises the codons UAA and UAG. RF2 recognises UAA and UGA. RF3 helps either RF1 and RF2 to carry out the reaction.

99. Translation in Eukaryotes:- INITIATION:- Eukaryote have atleast 9 initiation factor. Eukaryotic initiator t RNA does not become formylated as in prokaryotes. ELONGATION:- The factors eEF1a,eEF1b,eEFz are involved in elongation.

100. TERMINATION:- Eukaryotes have only one release factor eRF which recognize all the stop codons .

101. REFRENCE:- BIOTECHNOLOGY FUNDAMENTALS BY FIRDOS ALAM KHAN