Nature structure and replication of genetic material
- 1. Nature structure and replication of genetic material Submitted by J. Venkata Yashwanth TAM/2019-042 ACHARYA N G RANGA AGRICULTURAL UNIVERSITY S.V.Agricultural college, Tirupati Dept. of Genetics and Plant breeding
- 2. What is genetic material???? Have you people ever wondered why we look similar to our parents and their characters? Why some diseases are called inherited diseases and how do they inherit? How characters are transferred from parent to offspring's? The answer is very simple it is due to genetic material Now the real question is what is that genetic material exactly.
- 4. Properties of genetic material Genetic material is a chemical of which are genes composed Replicated with high fidelity Inherited with high degree of precision Able to express itself on development of character It should store high variable information for gene function
- 6. Identification of genetic material After discovery of Nuclein by Mischer there was hunt to know what is the genetic material Experiments of Grifth Experiments by Avery, Mac leod and Mc carty Experiments of Hershey and Chase
- 7. Experiments by Grifth Grifth discovered the phenomenon of transformation through s.pneumonieae Smooth colonies - Strain IIIS – virulent Rough colonies – Strain IIR – avirulent Live strains of IIR were injected into mice all mice survived Live strains of IIIS were injected into mice all mice died Heat killed IIIS were injected all mice survived Heat killed IIIS+IIR were injected together then all the mice died
- 9. Experiments by Avery Mc leod and Mc carty They carried out experiments invitro conditions in 1944 When live IIR and heat killed IIIS cells were plated on colony with Anti-IIR gave smooth colonies They used Proteases DNases and RNases to find out what is genetic material From the experiments carried out thy decided that DNA is the actual genetic material
- 11. Experiments of Hershey and chase From this experiment DNA was universally accepted as genetic material They used T2 bacteriophage which infects bacteria The head coat of bacteriophage is made of proteins and nucleic material is made up of DNA They labelled DNA with P32 protein with S35 When P32 phage's were cultured in E.coli for 10 min and centrifuged ghosts particles were normal and E.coli had P32 Progeny phage's contained P32 DNA
- 12. In other experiment they cultured S35 phage's with E.coli for 10 min The ghosts that were outside consisted of s35 while progeny was normal By these observations they came to conclusion that DNA is the ultimate genetic material
- 14. RNA as genetic material In several viruses like TMV the DNA is absent in them it again lead to confusion of genetic material In 1956 Gierer and Schramm and Frankel-Cornat independently demonstrated RNA functions as genetic material The proteins and RNA from were separated chemically and injected into Tobacco plant separately Plants injected with RNA were showing prevalent symptoms
- 15. Frankel-Cornat and Singer constructed two types of hybrid virus particles by mixing two different strains Type A has RNA of Strain A and protein from strain b and type b has RNA of strain B and protein of strain A When plants were injected with type a virus they showed symptoms similar to strain A When plants were injected with type b virus then they showed symptoms similar to strain b By this they concluded that it is RNA that is acting as genetic material but not proteins
- 16. Constituents of nucleic acid Chemical analyses of nuclei acids shown that they are basically composed of three components 1) Phosphoric acid 2) Pentose sugar 3) Organic base
- 17. Phosphoric acid(H3po4) – it has three reactive -OH groups among which two are involved in phospho diester bond with pentose sugar It makes bonding with 5’c of one and 3’c of neighbouring pentose molecules Pentose sugar – it is of two types Ribose sugar and deoxyribose sugar Deoxy ribose sugar differs in O atom at 2’c Always 5’ and 3’ carbons participate in phospho diester linkage and 1’ makes bonds with organic bases
- 18. Organic bases They are heterocyclic compounds which contain nitrogen so they are also called nitrogen bases They are of two types :- purines and pyrimidines Pyrimidine bases :- are 6 ringed pyrimidine structures similar to benzene ring with N- at 1 and 3 carbons and O- group at 2c position N at 1 participates in bond with pentose sugar In cytosine –NH2 group is present at 4c In uracil – O group is present at 4c Thymine – it is 5 methyl uracil
- 19. Purine bases They are two rings one 5 membered and one 6 membered N is present at 7th and 9th bases and 9 N participates in bod with pentose sugar Adenine – has –NH2 group a 6c Guanine – has –O group at 6c and –Nh2 at 2C N bases are have keto and enol tatuo-merisation but mostly they exist in Keto tautomeric forms
- 21. NUCLEOSIDES Nitrogen bases are linked with pentose sugar by covalent bonds between 1’c of sugar and 1c or 9c of nitrogen base This reaction release one molecule of h2o When ribose combines with N base it forms Ribo-nucleosides E.g.:- adenosine guanosine cytidine uridine When deoxyribose sugar combines with N base it forms deoxy- nucleosides E.g.:- deoxy-adenosine deoxy-guanosine deo-cytidine, thymidine
- 22. NUCLEOTIDES When phosphate group of phosphoric acid is attached to nucleoside or deoxy-nucleoside it forms nucleotides or deoxynucleotides 5’nucleotide – it has free -OH on 3’C 3’nucleotide – it has free -OH on 5’c 5’adenylic acid 5‘guanylic acid 5’cytidilic acid 5’uridylic acid are examples of ribotides 5’deoxyadynelic acid 5’deoxyguanylicacid 5’deoxycytidilic aid are example of deoxyribotides
- 24. The primary structure of DNA DNA is generally double stranded and each strand is multimer of deoxy- ribotides and called as polynucleotide Two nucleotides are joined each other with phospho diester bond Phosphodiester bond is represented as 5’C-O-P-O-C3’ The 5’C of one nucleotide and 3’C of another nucleotide have free –OH grp which is used to add deoxy-ribotides and form polynucleotide In polynucleotides N base sticks out of sugar phosphate backbone
- 25. THE DNA DOUBLE HELIX The DNA double helix model was given by Watson and crick in 1953 Before Watson and crick there were few scientists who works on model of DNA The ability of deoxy-ribotides to produce polynucleotides was discovered by Levene But he proposed that polynucleotides occurred in a repeated tetranucleotide sequence e.g.:- AGTC AGTC …….
- 26. THE CHARGAFF RULES Chemical analyses by Chargaff shown that Number of purines and number of pyrimidines are equal in number In DNA quantity of A is equal to quantity of T and quantity of g is equal to quantity of c In DNA the number of bases containing –NH2 groups and number of bases contain keto groups are in equivalence The above stated ones are called Chargaff rules
- 27. X ray crystallography by Franklin The x ray crystallography of purified DNA by franklin showed that It is a multi-stranded with diameter of about 22A The groups were spaced at 3.4A There was occurrence of repeating unit every 34A Watson and Crick made use of both Chargaff rules and x ray crystallography by franklin in constructing DNA double helix
- 28. Features of DNA double helix DNA molecule is made up of two polydeoxy-ribotide strands Two strands of DNA are oriented in anti parallel direction Polynucleotide strand is composed on deoxy-ribotides joined by phospho diester linkage The two strands of DNA are complementary (A=T and G=C) If A≠T and G≠C then the DNA is single stranded Adenine of one strand pairs with thymine of another strand with two hydrogen bonds Guanine of one strand with cytosine of another strand with three hydrogen bonds
- 31. This type of pairing is called complementary base pairing and base pairs are called Watson crick base pairs Hydrogen bond is weaker bond and denaturation occurs when ph. is raised in alkaline range Two strands are coiled in right hand helix forming DNA double helix The diameter of this helix is 20A while pitch is 34A There are 10 bases in a turn at equal spacing of 3.4A In aqueous medium the non polar groups of bases make hydrophobic interaction keeping DNA together
- 32. If we dissolve the DNA in distilled water two strands repel each other due to negative charge and in aqueous solution these negative charges are neutralized by Na+ ions and keeping DNA together During replication DNA uncoils and single strand individually participates n replication which avoids error If there is any error in base pairing during replication then there is chance of occurring of mutations The DNA structure explained by Watson and crick is B type and there are three more forms of DNA found in living organisms Apart from B-DNA there are other forms of DNA occurring in living organisms
- 33. Forms of DNA The different forms of DNA are A,B,C and Z From above forms only Z DNA shows left hand coiling the remaining three right hand coiling Dehydrated DNA occurs in form of A DNA it is unusual that naïve DNA occurs in this B form only DNA:RNA heteroduplexes and RNA double strands occur in this A form B DNA has major and minor grooves and less negative charge C DNA is tightly wounded when compared to z DNA
- 36. DNA replication
- 37. Central dogma
- 38. Replication of genetic material There are three methods of replication 1)Dispersive replication 2)conservative replication 3)semi-conservative method of replication The first two are not accepted as there was no practical proof but semi conservative model was proven by experiments of Melson and Stahl by their experiments on E.coli
- 39. A) Semi conservative replication B) Conservative replication C) Disruptive replication
- 40. Semi conservative model of replication It was proposed by Watson and crick at time of DNA double helix The main features of this model are:- Progressive separation of strands Complementary base pairing Formation of phosphodiester bonds with neighbouring single strands Each daughter DNA has one strand new and one strand parent DNA Sequence of baes on newly synthesized strand is dictated by template strand from parent
- 41. Evidence for semi conservative replication It was given by Meselson and Stalh in1958 They used two isotopes of nitrogen, the common 14N and heavier 15N to label nitrogen in DNA. The density of N15 was higher (1.724g/cm2 while n14 was less (1.710g/cm2) They used caesium chloride(CsCl) equilibrium density gradient centrifugation as their sedimentation method When DNA replicates, it incorporates labeled nitrogen atoms (either 14N or 15N) into DNA molecule, as nitrogen is an essential chemical component of DNA. Therefore nitrogen was chosen for labeling of DNA molecule.
- 42. They grew a culture of E.coli bacteria in a medium that contained heavier 15N for 14 bacterial generations, which was long enough to create a population of bacterial cells that contained only the heavier isotope. Next, they used the medium containing only 14N labeled ammonium salts as the sole nitrogen source. So, from that point onward, every new strand of DNA would be built with 14N than 15N. Then E.coli were given one generation time and isolated DNA was extracted from them and subjected to centrifugation These light and heavy strands can be separated by density gradient centrifugation in heavy salt solution at 30000 to50000 rpm for 18 to 72 hours
- 44. In first generation we can see that all DNA is intermediate In second generation DNA half of the strands were intermediate and half were normal In each succeeding generation the quantity of intermediate DNA was reduced to half All the above results shown that DNA is replicated by semi conservative method
- 47. Auto radiography of replicating E.coli chromosome At replication fork one DNA molecule occurs to be branched due to progressive separation of two strands The presence of replication fork was first shown by J.Carrins in E.coli He cultured E.coli cells in presence of 3H-thymidine He isolated the DNA from them on a filters and affixed on a glass with photograph emulsions sensitive to beta rays He observed 0-shaped structures and proposed that semi conservative replication starts at point called origin and proceeds in uni direction
- 48. Replicon REPLICON: Unit of DNA, capable of DNA replication independent of other segments of same DNA molecule. Each replicon has 1. Origin 2. Terminus Each replicon fires only once in a cell division Incase of prokaryotes and viral chromosomes, contains single replicon/ chromosome and incase of eukaryotes multiple replicons are seen T7 phages which has 2 replicons but one is non functional in presence of first In prokaryotes segregation and replication are same
- 49. origin It is sequence of replicon which supports initiation of replication In E.coli is identified as genetic loci ORI C It helps in initiation of replication control of frequency of events and segregation of replicated chromosome There are rich in A:T ration helpful in unwinding The length of E.coli replicon is only 245bp long In yeast we have autonomously replicated sequences (A) that is bounded to orc(origin recognition complexes) which helps in initiation Three elements (B1 B2 B2) are present I upstream and any two B and A sequence is necessary to start replication
- 50. In eukaryotes, no. of replicons per chromosome varies with species Yeast-500 Drosophila-3500 Vicia faba-35000 length of eukaryotic replicon varies with species to species ~40kb in yeast ~100kb in animals ~300kb in vicia rate of replication rate prokaryotes: 50,000bp/min eukaryotes: 2000bp/min No. of detectable replicons vary with developmental stage ,cell or tissue type. only 15% of replicons seem to be active at one moment.
- 52. Terminus Prokaryotic replicon's have two terminus in general E.coli has terA terC terD terE Termination requires tus gene product which recognizes ter sequence and Tus gene has contra helicase activity Replication fork moves 10 times faster than the replication In prokaryotes generally replication and transcription takes place simultaneously
- 53. REPLICATION INVOLVES 3 MAIN EVENTS 1 .Initiation 2. Elongation 3. Termination
- 55. Replication in prokaryotes INITIATION: DNA replication begins from origin. In E coli, replication origin is called OriC which consists of 245 base pair and contains DNA sequences that are highly conserved among bacterial replication origin. Two types of conserved sequences are found at OriC, three repeats of 13 bp (GATRCTNTTNTTTT) and four/five repeats of 9 bp (TTATCCACA) About 20 molecules of dna A proteins binds with 9 mer repeats along with ATP which causes DNA to wraps around dna A protein forming initial complex. The dna A protein and ATP trigger the opening of 13 mer repeats forming open complex.
- 58. Two copies of dna B proteins (helicase) binds to 13 mer repeats. This binding is facilitated by another molecule called dna C. The dna B-dna C interaction causes DNA B ring to open which binds with each of the DNA strand. The hydrolysis of bound ATP release DNA C leaving the DNA B bound to the DNA strand. The binding of helicase is key step in replication initiation. dna B migrates along the single stranded DNA in 5’-3’ direction causing unwinding of the DNA. The activity of helicase causes the topological stress to the unwinded strand forming supercoiled DNA. This stress is relieved by the DNA topoisomerase (DNA gyrase) by negative supercoiling. Similarly, single stranded binding protein binds to the separated strand and prevents reannealing of separated strand and stabilize the strand. The DNA polymerase cannot initiate DNA replication. So, at first primase synthesize 10±1 nucleotide (RNA in nature) along the 5’- 3’ direction. In case of E.coli primer synthesized by primase starts with ppp-AG-nucleotide. Primer is closely associated with dna B helicase so that it is positioned to make RNA primer as ssDNA of lagging strand.
- 60. 2. ELONGATION: I. Leading strand synthesis: Leading strand synthesis is more a straight forward process which begins with the synthesis of RNA primer by primase at replication origin. DNA polymerase III then adds the nucleotides at 3’end. The leading strand synthesis then proceed continuously keeping pace with unwinding of replication fork until it encounter the termination sequences. II. Lagging strand synthesis: The lagging strand synthesized in short fragments called Okazaki fragments. At first RNA primer is synthesized by primase and as in leading strand DNA polymerase III binds to RNA primer and adds dNTPS. On this level the synthesis of each okazaki fragments seems straight forward but the reality is quite complex
- 62. Mechanism of Lagging strand synthesis The complexity lies in the co-ordination of leading and lagging strand synthesis. Both the strand are synthesized by a single DNA polymerase III dimer which accomplished the looping of template DNA of lagging strand synthesizing Okazaki fragments. Helicase (Dna B) and primase (Dna G) constitute a functional unit within replication complex called primosome. DNA pol III use one set of core sub unit (Core polymerase) to synthesize leading strand and other set of core sub unit to synthesize lagging strand. In elongation steps, helicase in front of primase and pol III, unwind the DNA at the replication fork and travel along lagging strand template along 5’-3’ direction. DNA G primase occasionally associated with DNA B helicase synthesizes short RNA primer. A new B-sliding clamp is then positioned at the primer by B-clamp loading complex of DNA pol III.
- 64. When the Okazaki fragments synthesis is completed, the replication halted and the core sub unit dissociates from their sliding clamps and associates with new clamp. This initiates the synthesis of new Okazaki fragments. Both leading and lagging strand are synthesized coordinately and simultaneously by a complex protein moving in 5’-3’ direction. In this way both leading and lagging strand can be replicated at same time by a complex protein that move in same direction. Every so often the lagging strands must dissociates from the replicosome and reposition itself so that replication can continue. Lagging strand synthesis is not completes until the RNA primer has been removed and the gap between adjacent Okazaki fragments are sealed. The RNA primer are removed by exonuclease activity (5’-3’) of DNA pol-I and replaced by DNA. The gap is then sealed by DNA ligase using NAD as co-factor.
- 65. Termination: Eventually the two replication fork of circular E. coli chromosome meet at termination recognizing sequences (ter). The Ter sequence of 23 bp are arranged on the chromosome to create trap that the replication fork can enter but cannot leave. Ter sequences function as binding site for TUS protein. Ter-TUS complex can arrest the replication fork from only one direction. Ter-TUS complex encounter first with either of the replication fork and halt it. The other opposing replication fork halted when it collide with the first one. This seems the Ter-TUS sequences is not essential for termination but it may prevents over replication by one fork if other is delayed or halted by a damage or some obstacle. When either of the fork encounter Ter-TUS complex, replication halted. Final few hundred bases of DNA between these large protein complexes are replicated by not yet known mechanism forming two interlinked (catenated) chromosome.
- 67. Initiation The first steps is the formation of pre-initiation replication complex (pre- RC). It occurs in two stage. 1st stage requires, there is no CDK activities. It occur in early G1 phase. It is known as licensing but licensed pre-RC cannot initiate replication at G1 phase. 2nd stage is binding of ORC (origin recognition complex). The replication begins with binding of ORC to the origin. ORC is a hexamer of related protein and remains bounded even after DNA replication occurs. Furthermore ORC is analogue of prokaryotic DNA A protein. After binding of ORC to origin, cdc6/cdc18 and cdtl coordinate the loading of MEM (mini chromosome maintenance) to origin. MEM complex is thought to be major eukaryotic helicase. After binding of MEM complex to pre-RC, cdtl get displaced. Then DdK phosphorylates MEM, which activates its helicase activity. Again DdK and CdK recruit another protein called cdc45 which then recruit all the DNA replicating protein such that the origin get fired and replication begins.
- 68. 2. Elongation: DNA polymerase δ synthesizes and adds dNTPs at 3’ end of RNA primer. The leading and lagging strands are synthesized in the similar fashion as in prokaryotic DNA replication. 3.TERMINATION: At the end of DNA replication the RNA primer are replaced by DNA by 5’-3’exonuclease and polymerase activity of DNA polymerase ε. Exonuclease activity of DNA polymerase removes the RNA primer and polymerase activity adds dNTPs at 3’-OH end preceding the primer. In case of bacteria, with circular genome, the replacement of RNA primer with DNA is not a problem because there is always a preceding 3’-OH in a circular DNA.
- 69. But in eukaryotic organism with linear DNA, there is a problem. When RNA primer at 5’ end of daughter strand is removed, there is not a preceding 3’-OH such that the DNA polymerase can use it to replace by DNA. So, at 5’ end of each daughter strand there is a gap (missing DNA). This missing DNA cause loss of information contain in that region. This gap must be filled before next round of replication. For solving this end replication problem; studies have found that linear end of DNA called telomere has G:C rich repeats. These sequence is known as telomere sequence. These repeats of telomere sequence is different among different organisms. Telomere in human cell consists of repeats of TTAGGG/AATCCC. Each species has its own species specific telomere repeats. These telomere sequence don't codes anything but it is essential to fill the gap in daughter strand and maintain the integrity of DNA.
- 71. Telomere replication: end replication problem in Eukaryotic DNA There is an enzyme found in eukaryotic cell called telomerase. Telomerase is a DNA polymerase (RNA dependent DNA polymerase) which adds many copies of telomere sequence at 3’- OH end of template strand. Like other DNA polymerase, telomerase also adds deoxyribonucleotide at 3’-OH end. Unlike other DNA polymerase, telomerase adds DNA at 3’-OH end of parent strand not at the daughter strand and also it synthesizes the same sequences over and over in absence of template strand. First telomerase binds to 3’-OH end of parent strand by hybridization between its AACCCCAAC RNA sequences and TTGGGG DNA sequences (telomere sequences of Tetrahymena). The telomerase adds TTG at 3’ end of parent strand. After adding TTG sequences, telomerase translocate along 5’-3’ end of parent strand. Now the telomerase adds GGGTTG to 3’ end by using its CCCAAC sequence. Again telomerase translocate and adds GGGTTA
- 72. This process is continued for many time. The parent strand become more longer than daughter strand. Now RNA polymerase (PRIMASE) synthesize RNA primer by copying the parent strand in 5’-3’ direction using telomere sequence as template. The DNA polymerase can now extend the primer in 5’-3’ direction by adding deoxyribonucleotide to 3’ end. The primer is now removed and it won’t be replaced because it is an extra sequence added by copying telomere sequence. Finally the integrity of daughter strand is maintained.
- 74. Enzymes or proteins involved in DNA replication DNA replication involves several proteins and enzymes Many of these are identified by study of mutants In E.coli DNA E DNA N DNA X and DNA Z Code for four out of seven polypeptides of complete DNA polymerase III enzyme Genes involved in DNA replication are studied by isolating series of temperature sensitive mutants called DNA mutants They cease DNA replication when temperature is increased to 42*degrees which are of to types Quick stop DNA mutants Slow stop DNA mutants
- 75. DNA polymerase Also called DNA replicase that synthesizes new strand of DNA Its activity was first observed by Kornberg in 1956 It cannot initiate synthesis but adds nucleotides to free 3- OH on primer end It has property of proof reading They are aging classified into prokaryotic and eukaryotic polymerases
- 76. Prokaryotic polymerases DNA polymerase I or korenberg enzyme It has 3 function )5’—3’ polymerase 2)5’—3’ exonuclease 3) 3’—5’ exonuclease Exonuclease activity is observed in different regions 3’—5’ exonuclease is essential in proof reading and 5’—3’ exonuclease removes DNA hat is affected by uv light chemicals etc. It has unique property to initiate replication at a nick in strand of DNA duplex this property of nick translation DNA polymerase II has same activity but mainly act as DNA repair
- 77. DNA polymerase III It has 5’—3’ polymerase and 3’—5’ exonucleases activity and responsible for in vivo replication In eukaryotes γ form of DNA polymerase is present in mitochondria Pol α primes the replication in both the strands δ and ε form of polymerases are terminatory in nature Pol β is monomer and carries out high fidelity excision repair
- 78. Primase It catalyses synthesis of RNA primers Primases are rna polymerases that synthesis 11-12 base pairs Primases of E.coli and some viruses have Dna G a single polypeptide that associates with plication complex In eukaryotes the same function is performed by pol α and extends the primers as DNA strand then it is replaced by pol δ and pol ε
- 79. Polynucleotide ligase This enzyme important in DNA replication and repair Its's forms phosphodiester linkage between ‘phosphoryl group of one nucleotide and -3OH group of immediate nucleotide DNA ligase seals the nick that is left by DNA polymerase I during DNA repair and among okazaki fragments Endonucleases :- restriction endo nuclease produces cut in DNA at only specific bases E.g.:- phage θX174 It introduces a nick in surrounding DNA damage leaving free 5’ end for DNA polymerase I to cleave off the damaged
- 80. Helicase An enzyme that separate's two DNA strands It is multimer which has two conformations one that binds with DNA duplex and one that binds with single stranded DNA 1 ATP molecule is hydrolysed for unwinding 1 base pair Further it may be 3’—5’ helicase or 5’—3’ helicase Single stranded binding proteins (SSB) proteins SSB proteins binds to single stranded DNA and prevents forming duplex It binds to DNA as replication fork advances and mutants that lack ssb function are defective in repair and recombination Pilot proteins are synthesized by virus DNA that decides to go replication or transcribed by rna
- 81. Replication of genetic rna Direct replication of rna In many viruses like polio virus influenza viruses direct replication of rna by rna primed rna synthesis It is RNA dependent rna polymerase It requires a rna template mg++ ions and triphosphate ribo- nucleotides The replication of ss rna produces both + and – strands but only the + strands are packaged into virions
- 82. Replication via DNA In some viruses called as tumour inducing virus rna first synthesis complementary DNA which is transcribed into rna It is done by rna dependent DNA polymerase or reverse transcriptase This DNA helps for integration of viral genetic material into host Some viruses can remain as pro virus or cause cancerous cells Such virus are called oncogenic viruses