MUNI SCI Bi4025en Molecular Biology Mgr. Jiří Kohoutek, Ph.D. 1 Department of Experimental Biology Lecture 3 • Molecular structure and replication of procaryotic and eukaryotic genomes. 2 Department of Experimental Biology MUNI SCI Order versus chaos • Cells maintain a high degree of orderliness in a disordered universe. • This property is largely due to their capabilities to duplicate their genetic information with great precision. • The same genetic information divided into daughter cells ensures their similarity. • Reproduction of the chemical information in DNA- replication. 3 Department of Experimental Biology MUNI SCI Replication of nucleic acids • Replication is a biological process of duplicating or producing an exact copy, such as a polynucleotide strand of DNA. Creation of replicas (copies). • It is a molecular process taking place in dividing cells, by which the DNA creates a copy of itself. • Human speed of replication: 3 000 nucleotides/min. • Bacterial speed of replication: 30,000 nucleotides/min. Accuracy: one error per 109 embedded nucleotides. 4 Department of Experimental Biology https://www.biologyonline.com/dictionary/replication MUNI SCI Stability of the genome • Individual survival requires a high degree of genetic stability. • If the change occurs in the DNAand the repair mechanisms do not correct it, then, it becomes permanent - referred to as mutation. • If the mutation is located within some area of DNA important for life, it can cause disease of the organism or even its death. 5 Department of Experimental Biology MUNI SCI Frequencies of mutations in bacteria LOW Can be determined experimentally: • E. coli in laboratory conditions divides once every 30 minutes. • One cell in less than a day creates a population of several billion cells. • Only a fraction of cells in which a mutation has occurred in a certain nonessential gene can be found . • Conclusion: A gene of average size (about a thousand nucleotide pairs) is affected approximately 1x in 106 bacterial generations. • Mutation rate in bacteria: about 3 nucleotide changes per 1010 nucleotides per 1 cell generation. 6 Department of Experimental Biology MUNI SCI Frequencies of mutations in humans • The mutation rate adjusted to one round of DNA replication is extremely low in both bacteria and humans. Table 6-1 Error Rates US Postal Service on-time delivery of local first-class mail Airline luggage system A professional typist typing at 120 words per minute Driving a car in the United States DNA replication (without mismatch repair) 13 late deliveries per 100 parcels 1 lost bag per 200 1 mistake per 250 characters 1 death per 104 people per year 1 mistake per 107 nucleotides copied DNA replication (including mismatch repair) 1 mistake per 109 nucleotides copied Department of Experimental Biology Principles of DNA replication • Strands in DNA helix duplexes are complementary: after separation, each of them can serve as template for the synthesis of new strand. • New strands are created through gradual integration of nucleotides based on base pairing rules. • DNA replication is catalyzed by an enzymes. • Once replication is finished, each template strand is paired with a newly synthesized strand. Template strand 8 Department of Experimental Biology Alberts etal.: Molecular biology of the cell. Garland Sci. 2008, 2015 G Nucleotide precursor about to bind T = A ÄHU- C = G t Base pairing by H bonds Incoming bases forming new strand MUNI SCI Replication occurs in the S-phase of cell cycle • Replication cannot occur repeatedly within one cell cycle (similar to other phases). • DNA must be replicated before the cell divides in Mitosis to ensure the new cells have DNA. • DNA is replicated during the S-phase of the cell cycle. 9 Department of Experimental Biology https://quizizz.com/admin/quiz/606682e1825246001f488abd/dna-structure-function-and-replication-basics MUNI SCI Models of DNA replication Conservative model • In the conservative model, the parental molecule directs synthesis of an entirely new double-stranded molecule, such that after one round of replication, one molecule is conserved as two old strands. • This is repeated in the second round. i ^ Parental After first replication cycle After second \ _ : »• replication \ cycle 10 Department of Experimental Biology https://www.mun.ca/biology/scarr/iGen3_03-01.html MUNI SCI Models of DNA replication In the dispersive model, material in the two parental strands is distributed more or less randomly between two daughter molecules. In the model shown here, old material is distributed symmetrically between the two daughters molecules, yet other distributions are possible. Dispersive model ^ ^ Parental After first replication cycle *y After second V ^ v ^ replication v ^ I ^ 11 Department of Experimental Biology https://www.mun.ca/biology/scarr/iGen3_03-01.html MUNI SCI Models of DNA replication Semiconservative model In the semi-conservative model, the two parental strands separate and each makes a copy of itself. After one round of replication, the two daughter molecules each comprises one old and one new strand. Note that after two rounds, two of the DNA molecules consist only of new material, while the other two contain one old and one new strand. i Parental After first replication cycle ~ After second I ^ I ^ replication v ^ t ^ ©2010 Pearson Education. Inc. 12 Department of Experimental Biology https://www.mun.ca/biology/scarr/iGen3_03-01.html MUNI SCI DNA replication is semi-conservative process In 1958 the semi-conservative model of replication, proposed by Watson and Crick in 1953, was proved. Evidence based on the study of DNA density after marking with heavy nitrogen N15. Matthew Meselson Franklin Stahl 14 generations of growth E.coli X. E COli CsCL Serm-conservative Replication Generation C Q — 100% 25% ; ■2'. r O 20 40 60 80 Time (min.) 13 Department of Experimental Biology https://www.thoughtco.com/dna-versus-rna-608191 MUNI SCI Start 15N-containing medium Continue growing first generation in 14N medium Replication cycle 1 Continue growing Replication cycle 2 Continue growing Replication cycle 3 DNA in CsCI gradient DNA composition <3 J 2 I5N-1SN (heavy) ,5 DNA <5 . ? >5N-1"N ■>^J (intermediate r > density) ^ DNA 14N-14N DNA ■5 <^J (intermediate £ s density) DNA !4N-14N UN-14N "N—'*N '5N-14N Department of Experimental Biology © 2010 Pearson Education. Snc. Photographs of DNA bands I I II I Ä £ %%% -p y> t %%% Densitometrie scans (Tl 5 Ü i5 %%% -? y -p <&> 'S 'S 5. $ DNA replication is semi-conservative process Fuuu »-3. (Left) Muihe* Mc-hnn |Courtet> of M. Mcacbon ) https://www.mun.ca/biology/scarr/iGen3_03-02.html MUNI SCI DNA replication is semi-conservative process 1) Conservative: o no molecules with hybrid density. 2) Semi-conservative: o there is hybrid density. 3) Dispersive: o gradually decreasing hybrid density. Conservative Semi-con servative J) Dispersive CsCI ultracentrifugation 15 Department of Experimental Biology https://bio.libretexts.org/Bookshelves/Cell_and_Molecular_Biology/Book%3A_Cells_-_Molecules_and_Mechanisms_(Wong)/07%3A_DNA/7.02%3A_Semi-Conservative_DNA_Replication MUNI SCI Basic characteristics of DNA replication DNA replication is Semi-conservative. Both dsDNA strands serve as templates. The result of DNA replication is a double helix containing one original and one newly synthesized strand. Each parental strand remains preserved. The original strands remain intact for many generations. REPLICATION REPLICATION III? an A A A ^REPLICATION y\ /\ X 7v %%%%%%%% lllllli Department of Experimental Biology MUN SCI DNA replication - biochemical view • Replication is a process of producing an exact copy of polynucleotide strand of DNA. • New strands are created through gradual integration of nucleotides based on base pairing rules. • DNA polymerase is the key enzyme catalyzing the synthesis of the DNA strand. 5' r / p - A p p — G OH / , 3 / □ / ^ < 3' OH - ■ Pv " E < / / \ \ P / p T »!!»!: .....»■• A / P / / G / p P / > primer / p / P / , L / G / _ 5' HO P S 5' 17 Department of Experimental Biology MUNI SCI Basic characteristics of DNA replication • DNA replication is Semi-discontinuous. • Continuous Replication: o It occurs on the leading strand. o Progresses from 5' end to 3' end in the direction of the replication fork, o In vivo no need for RNA primer. • Discontinuous Replication: o It progresses opposite to the leading strand on the lagging strand from 3' end to 5' end. o It starts somewhere in the DNA and away from the replication fork. o Needs RNA primers. DNA replication fork leading strand 3' tagging strand MUNI 18 Department of Experimental Biology https://www.quoraxom/How-does-a-continuous-DNA-replication-differ-from-a-discontinuous-one _ _ O 0 J. Okazaki's fragments • Explorers: • Reiji Okazaki (1930-1975) and his wife Tsuneko Okazaki H933). • Nagoya University, Japan. • Reiji died prematurely of leukemia- The result of the radiation exposure of the Hiroshima bombing. • Tsuneko - first woman professor in Nagoya University. 19 Department of Experimental Biology MUNI SCI Basic characteristics of DNA replication Okazaki fragments 5' 3' 3' 5' direction of fork movement leading-strand template of left-hand fork lagging-strand template of right-hand fork lagging-strand template of left-hand fork leading-strand template of right-hand fork DNA polymerase catalyzes the growth of the new DNA strand in 5' to 3' direction. Thus DNA replication is asymetrical, due to continuous and discontinuous synthesis of new DNA strand at the at the tip of replication fork. 20 Department of Experimental Biology https://slideplayer.com/slide/13166744/ MUNI SCI Beginning of DNA replication Initiation of DNA replication takes place in specific places - „origins of replication". From the beginning DNA replication takes place in both directions (always in the direction of 5'- 3'). Each beginning ensures replication of a stretch of DNA called a „replicon". In bacteria and viruses, there is usually 1 origin per chromosome (prokaryotic chromosomes form a single replicon). In large eukaryotes chromosomes, there are many origins of replication (many replicons). A. £. coti chromosome 500,000 bp' B. Portion of eukaryotic chromosome Origin Origin Origin eo.ooo bp Department of Experimental Biology https://basicmedicalkey.com/s-phase-and-dna-replication/ S Beginning of DNA replication • Once DNA is released at the ori (origin of replication) site by the action of helicases, the template strands are continuously separated and a replication bubble is formed. Replication proceeds from this point in both directions and a structure of "Y- shape" is formed and is called replication fork. • Movement of the replication fork is coordinated with metabolic processes responsible for the synthesis of dNTPs. i-1 1 |_m 22 Department of Experimental Biology MUNI SCI Beginning of DNA replication Free nucleotides DNA polymerase , Old (parental) strand acts as a O 2013 Poarwjn EducrtlOn inc 23 Department of Experimental Biology https://quizlet.com/ca/246045954/dna-molecules-of-heredity-diagram/ MUNI SCI Replisome • The replisome is a large protein complex that carries out DNA replication, starting at the replication origin. • It contains several proteins with enzymatic activities: o Helicase o Primase o DNA polymerase o Exonuclease o Topoisomerase 24 Department of Experimental Biology http://dx.doi.org/10.1016/bs. Bacterial fork MUNI .2016.03.004 § C Parts of DNA replication complex • Template (matrix strand) = parent molecule. • Nucleotides (dNTP). • Primer = short oligoribonucleotide with free 3 -OH end. • Enzymes catalyzing the joining of nucleotides o Primase o Polymerase o Ligase. — ATCCGTGCTGCTTGCTTGAATACC 5' UAGGCACGA-3'OH---► RNA primer synthesized strand MUNI SCI 25 Department of Experimental Biology DNA replication phases Steps involved in DNA replication: Initiation Initiation J implicative DNA heltcase Elongation Termination Elongation J Positive Supercoils * resolved byTopo I orTopo II) Elongation Termination \ Convergence & Encounter A pre-Catenane ^ Disassembly & Gap filling H Catenane \( \o( J Decatenation by topo II 26 Department of Experimental Biology Nat Rev Mol Cell Biol. 2017 Aug; 18(8): 507-516. MUNI SCI • Prokaryotic DNA replication MUNI 27 Department of Experimental Biology _ _ T O 0 J. Initiation phase - origin of DNA replication • The specific sequence called oriC in bacteria is recognized by a DnaA factor. • Size 245 pb. • Present in the genome 1x. • Two types of repeating Sequences: o sequence 13 pb (repeated 3 times, rich in AT, place of loosening), o sequence 9 pb (repeated 4 times, binding region of proteins, which are necessary for the formation of a replication bubble). Bacterial replication origin 1 17 32 58 GATCTNTTTATTT — GATCTNTTNTATT 166 201 240 TTATACACAt TTTGGATAA TTATCCACA 28 Department of Experimental Biology https://basicmedicalkey.com/s-phase-and-dna-replication/ MUNI SCI Initiation phase - unwinding Dita A sites ů*»D*ůA+ATP- HUnriHF- B ATPN An initiator protein (product of the E. coli DnaA gene) binds to this origin and either directly or indirectly. DnaA promotes melting of the DNA duplex, giving the replication machinery access to two single strands of DNA. DnaB / helicase unwinds oriC (origin of replication) and extends the single stranded region for copying. 29 Department of Experimental Biology https://basicmedicalkey.com/s-phase-and-dna-replication/ MUNI SCI Initiation phase - binding of SSB proteins 13-mers Di>aA sites *»DnaA+ATP-" HUůríHF- B ■ complex ATP^ EM Single strand binding protein (SSB) binds to this single stranded region to protect it from breakage and to prevent it from renaturing. Other factors bind to the initiator, and their concerted action produces a wave of DNA replication proceeding outward in both directions along the DNA (a replication "bubble"). As the parental DNA is unwound by DNA helicases and SSB (travels in 5'-3' direction). 30 Department of Experimental Biology https://basicmedicalkey.com/s-phase-and-dna-replication/ MUNI SCI Initiation phase - DNA twisting issue DNA rotates in front of the replication fork due to unwinding of DNA helix. The unwinding of DNA leads to the formation of „positive supercoiled loops". • The resulting positive supercoiling (torsional stress) is relieved by topoisomerase I and II (DNAgyrase) by inducing transient single or double stranded breaks. Department of Experimental Biology Elongation phase - Leading strand Leading strand synthesis: • On the template strand with 3'-5' orientation, new DNA is made continuously in 5'-3' direction towards the replication fork. The new strand that is continuously synthesized in 5'-3' direction is the leading strand. • DNA polymerase III extends the RNA primer made by primase. DNA polymerase possesses separate catalytic sites for polymerization and Elongation phase - Lagging strand Lagging strand synthesis: • On the template strand with 5'-3' orientation, multiple RNA primers are synthesized at specific sites by primase (primosome complex). • DNA pol III synthesizes short pieces of new DNA (about 1000 nucleotides long) new DNA is in 5'-3' direction. • The new strand which is discontinuously synthesized in small, Okazaki's MUNI SCI Elongation phase - RNA primers degradation and ligation • DNA polymerase III synthesizes DNA for both leading and lagging strands. • After DNA synthesis by DNA pol III, DNA polymerase I uses its 5'-3' exonuclease activity to remove the RNA primer and fills the gaps with new DNA. • Finally DNA ligase joins the ends of the DNA fraaments together. Termination phase • The two replication forks meet approximately 180 degree opposite to oriC, as DNA is circular in prokaryotes. • There are several terminator sites - ter (typically 10 sites) which form termination zone. These sites arrest the movement of forks by binding to the terminus site-binding protein (Tus), an inhibitor of helicase (DnaB). • The ter sites are oriented such that the leftward fork can pass the first five ter sites it encounters (red arrowheads), but stalls at the five blue sites. Conversely, the rightward fork passes through the ter sites marked as blue arrowheads but stalls at the red sites. oriC leftward fork rightward fork Polar Fork Barriers Permissive orientation Termination zone Non-permissive orientation —*4 Fork 1 (arrives first and stalls at ter site C) II 35 Department of Experimental Biology Nat Rev Mol Cell Biol. 2017 Aug; 18(8): 507-516. MUNI SCI Termination phase 1. Fork convergence (TopolV) j 2. 3' flap generation ▼ 3' 1. Helicase dissociatiof 2. 3' flap removal 3. Gap filling 4. Okazaki maturation III ~ DNA pol I 1. Ligation ■ 2. Decatenation (not shown) \ 36 Department of Experimental Biology • In this way, forks can enter but not leave the termination zone. • Topoisomerase IV - TopolV induces fork convergence and 3'flap is generated. • The flap is normally degraded or remodeled and the gap is subsequently filled. • Polymerase I may use its 5' to 3' exonuclease activity to remove the RNA primer of the last Okazaki fragment. • Once replication is complete, the two double stranded circular DNA molecules (daughter strands) remain interlinked. Topoisomerase II unlink these molecules. „ „. MUNI Nat Rev Mol Cell Biol. 2017 Aug; 18(8): 507-516. O 0 J. DNA Polymerase in prokaryotes • DNA polymerases are a group of enzymes that are used to make copies of DNA. • They are not used for initiating the synthesis of new strands, but in the extension of already existing DNA or RNA strands which are paired with a template strand. • They catalyze the formation of the phosphodiester bonds between nucleotides., extending the 3' end of the template strand. • Polymerase uses a magnesium ion (Mg2+) in catalytic activity to balance the charge from the phosphate group. 37 Department of Experimental Biology https://microbenotes.eom/dna-polymerase/#dna-polymerase-iii MUNI SCI DNA Polymerase in prokaryotes The addition of a nucleotide to a growing DNA strand forms a phosphodiester bond using the high-energy phosphate bond of hydrolysis and two distal phosphates known as pyrophosphate. • The 3' hydroxyl group (green oval) attacks the triphosphate group on the incoming nucleotide (violet circle). oh oh oh I J n ' _P-0-P-0- xh h oh h Base 1 Base 1 lb Base 2 oh OH I OH H Base 1 OH I -> o—j— 0H 0 Base 1 Base 2 S -och. oh ii I ° -p—oh + energy Base 2 A new phosphodiester bond is formed with the pyrophosphate. Pyrophosphate is cleaved to two phosphates and the energy is released from breaking down a the high energy phosphate group. oh h 38 Department of Experimental Biology https://chem.libretexts.org/Bookshelves/General_Chemistry/Book%3A_ChemPRIME_%2 8Moore_et_al.%29/20%3A_Molecules_in_Living_Systems/20.21%3A_DNA_Replication MUNI SCI DNA Polymerase in prokaryotes • DNA polymerases are very accurate in their mechanism with minimal errors of less than one error for every 107 nucleotides. • Some types of DNA polymerase have the ability to proofread and remove unmatched bases of nucleotides and correct them. • They also correct post-replication mismatches by monitoring and repairing the errors, by distinguishing mismatches of the new strand from the template strand sequences. 39 Department of Experimental Biology https://microbenotes.eom/dna-polymerase/#dna-polymerase-iii MUNI SCI DNA Polymerase in prokaryotes DNA polymerase Tamily Poll A Activity 5'-3ř polymerase 3''5' íxottuclease 5'-3' exjjnuclp^e PolA r-.i!iii ■■ . of ftioIecijIgsi' cell 400 Pol II B 51-3' polymerase 3 '-5' exorwjclease 50-75 350 • 1000 Biological functions In the cíH U N r ÉípJifi al i ů n, \ P N A ruplic 3 íi O n Ohazakl fragment Wbaekup DNA maturation, I polymerase}, □ NA repair / DNA repair, TLS Pol III c 5"-3' Pol IV V polymerase 5'' Pol V V \i polymere 150 - 250 1200-2500 TLS < 15 200 TLS 40 Department of Experimental Biology FEMS Microbiol Rev. 2012 Nov; 36(6): 1105-1121. MUNI SCI DNA Polymerase III LEADING STRAND • This is the primary enzyme that is used by prokaryotic cells in DNA replication. • Is able to synthesize long stretches of template DNA. • It is responsible for the synthesis of new strands , 5'- 3' orientation, by adding nucleotides to the 3'- OH group of the primer. • It has a 3-5' exonuclease activity hence it can also proofread the errors that may arise during DNA strand replication. 41 Department of Experimental Biology https://microbenotes.eom/dna-polymerase/#dna-polymerase-iii FEMS Microbiol Rev. 2012 Nov; 36(6): 1105-1121. MUNI SCI DNA Polymerase • DNA polymerase III is multisubunit complex. • Core polymerazition activity - a, z and 0 subunits. • (3 -dimer (clamp) prevents premature release of DNA-polymerases III from the template. Subunits of DNA polymerase III • a - polymerace - 5'- 3' • z - 3'- 5'- exonuclease activity • 0 - stimulation of £-subunit • Y, 5, - connection to (3-clamps (3 - clamp • t - dimerization of enzymes core units a. 42 Department of Experimental Biology - composition fi 20M John Wiley & Sons, trie. All rights reserved. MUNI SCI DNA Polymerase III - enzymatic activities RNA primer DNA strand added onto RNA primer 5' Base_ Pairs 3' HO /X/' Template DNA strand DNA polymerase III . / OH 5' • 5'- 3' synthesis of new strands from the RNA primer. 43 Department of Experimental Biology • 3' - 5' exonuclease activity in order to proofread the errors that may arise during DNA strand replication. Polymerase / Addition of INCORRECT base A C T A G I I « t I I 1 T T G C G A / Template DNA Removal of incorrect base 3' - 5' Exonuclease A C T A G TGATCCATTGCGA 3' Addition of CORRECT base A C T A G G MUNI SCI DNA Polymerase I • Its main function is excision repair of DNA strands from the 3-5' direction and the 5-3 direction, as an exonuclease. • Its role during replication is the removal of the ribonucleotides of the RNA primer, it moves along the 5-3' direction. • It also helps with the maturation of Okazaki fragments, which are short DNA strands that make up the lagging strand during DNA replication. 5'-3' exonuclease - RNA primer removal 44 Department of Experimental Biology https://pdb101 .rcsb.org/motm/3 MUNI SCI *A> Elongating DNA RNA primer DNA »1 rand polymer aw I for previous from upstream _» Iragment WW? Templalo DNA strand OH pair DNA Polymerase I • 1st ribonucleotide of RNA primer is triphospahted (NTP) (D) Next ON A nucleoid* added UNA nucleotide cleaved and eiecled WW OH • 5-3 exonuclease activity (C) Ejected RNA nuclvotidos OOOOO^O^y WWWvOooo^o Rcleajed RNA pnmor nuclooltda* (D) f T f f wwwwww$%wwww ho00 0.0.0 4.0 00 000 0 00.0.0.0.0.000 0 45 Department of Experimental Biology 5-3' polymerase activity https://pdb101 .rcsb.org/motm/3 • 3' - 5' exonuclease proofreading activity to correct the errors that may arise during DNA replication. MUNI SCI DNA polymerase III DNA polymerase I (A) 5'-*3 DNAsynth«ii Primer New DNA w \ XJJ 3 7 Template DNA poJyrocraj« Differences beetween DNA polymerase III and I • 5'- 3' synthesis of new strands DNA polymerase III DNA polymerase I (B) 3'—+5' exonucl«ue activity i i i i i i i i i i i i i>1 i i r...... 3* 5 • 3' - 5' exonuclease activity DNA polvrrttnj« revterce» řtt direction (C) 5'-»3' exonucleue *aMty Displaced nucleotides DNA polymerase I 46 Department of Experimental Biology 3" 111M1111111 irrn 1111M111111111 [ 5' - 3' exonuclease activity MUNI SCI Discovery of DNA polymerase In 1956 he isolated the DNA-polymerase I from E. coli, for the first time. 1959 - Nobel Prize in Physiology or Medicine for discovery of DNA synthesis. Roger Kornberg (*1947 Stanford university Arthur Kornberg (1918-2007) He isolated the DNA-polymerase III from E. coli, for the first time. 2006 - Nobel Prize in Physiology or Medicine for discoveries of mechanism of DNA replication. 47 Department of Experimental Biology MUNI SCI Unwinding of DNA double helix • The condition for the replication is the availability of unpaired nucleotides in the DNA strand = loosening of the double helix. • However, the double helix is stable (for denaturation the temperature close to the water boiling point is needed). • The opening of the double helix is enabled by 3 types of replication proteins: o DNA-helicase o Single strand binding (SSB) proteins o DNA-topoisomerase. 48 Department of Experimental Biology MUNI SCI DNA Helicase Unwinding of paraller DNA strands is a condition for their separation. The one turn of the Helicase subunits at the time. 1 turn of helicase -10 pb: 360° rotation for every 10 nucleotides. E. coli: replication rate of 30,000 nucleotides per minute. What is the speed of DNA rotation in turns/twists per minute? 3,000 turns/twists per minute. Eukaryotic (AAA+ motors) 3'to 5'translocations N-tier 5'to 3'translocations Bacterial (RecA motors) 49 Department of Experimental Biology https://www.pnas.Org/doi/10.1073/pnas. 1620500114 MUNI SCI DNA Helicase leading subunit helicase DNA polymerase • Six-compartment cylinders, that surround single-strand DNA. • Binds and hydrolyze ATPand thus move along single-strand DNA. • Once it encounters the double stranded region of DNA, Helicase continues its movement and separates bound strands from each other. 50 Department of Experimental Biology TTP (2'-deoxythymidine 5'-triphosphate) el_ife2015;4:e06562 DOI: 10.7554/el_ife.06562 MUNI SCI SSB proteins • Single-stranded DNA-binding proteins (SSBs) bind to single-stranded DNA (ssDNA) by wrapping the single DNA strand around the tetrameric protein core to protect it from degradation and prevent secondary structure formation. • They bind to DNA in a cooperative way (binding one monomer stimulates the bond of the other). • Bind tightly to the single stranded sections of DNA formed by the action of helicases, without blocking the bases, which thus remain available for pairing. Cooperative ssDNA binding SSB-SSB interactions SSB-partner binding 51 Department of Experimental Biology https://onlinelibrary.wiley.eom/doi/full/10.1002/pro.3115 MUNI SCI SSB proteins E. coli SSB is a homotetramer. Each monomer features a structured \ o DNA-binding domain (residues 1-112) o Long and disordered C-terminal tail (residues 116-177) containing a highly acidic tip. Arg in DNA binding groove between Lir and L \ 40 SSB35 I90-- 34 <=P 34. | *........ Acidic tip (168-177) Intrinsically disordered C-terminal domain (113-177) MUNI 52 Department of Experimental Biology NucleicAcids Res. 2017 Dec 1; 45(21): 12125-12139. OfT 0 U 1 Topoisomerase 53 Department of Experimental Biology Topoisomerase: • helps with prevention of DNA strand twisting - 'swivels'. Two types o Topoisomerase I - Break one strand only and then rejoin. o Topoisomerase II (Gyrase) - Break both strands and then rejoin. MUNI SCI Topoisomerase • DNA rotates in front of the replication fork by developing a helix. • Without the interruption of DNA strands by topoisomerases, the development of DNA leads to the formation of the positive supersuitical threads 54 Department of Experimental Biology MUNI SCI RNA primase After unfolding DNA at the site of ori by DNA-helicase the RNA-polymerase (primase) synthesizes special short sections of RNA. RNA-primers are complementary to the template strand. Primase (dnaG) synthesizes short stretches of RNA nucleotides, providing a 3'-OH group to which DNA polymerase can add DNA nucleotides in the direction of 5-3'. 10-60 nucleotides for prokarvotes. 10 nucleotides for eukarvotes. DNA 55 Department of Experimental Biology 3* PPP-5" SSB protein Parental DNA 4- A PriA DISPLACES SSB PROTEIN •I " "I PriA 4- .........■=■ B PRIMASE BINDS C PRIMASE MAKES SHORT RNA PRIMER Primosome ■ 1 1 1 ' 1 f RNA primer ""^^B k......= ■ Direction o! mo^emenl oi primostune > 3'-OH 5' RNA primer DNA MUNI SCI Primosome oriC • DNA-helicase (dnaB) and DNA-primase (dnaG) complex form Primosome. • Ensures the release of single strands from the dsDNA and the synthesis of RNA-primers. • Moves along a DNA molecule powered by ATP energy. 56 Department of Experimental Biology MUNI SCI Ligase • DNA-ligase corrects "notches" in the sugar-phosphate skeleton of DNA o DNA replication, o DNA Repair. • DNA-ligase is activated by ATP binding and temporarily joins the free 5 P at the notch site (P-P is released). • Release of ASF restores covalent bond in the strand. 5' P 3'OH AP P P P P " ~OH A P 5' ■ 5' I J J y m mm 1^1 —^—^—*■ I 11 ^ I ——- H I ^ ill A^JXJ-^l STEP 1 J.-_-J, -J, -\ STEP 2 _-J.-J-JL-J. AMP used released MUNI 57 Department of Experimental Biology Obi Replisome Curiwd Op*™ in structural Blotog/ 58 Department of Experimental Biology https://www.sciencedirect.eom/science/article/pii/S0959440X18300952#fig0025 MUNI SCI DNA replication in prokaryotes - overview 59 Department of Experimental Biology https://www.youtube.com/watch?v=bee6PWUgPo8 MUNI SCI Organization of dsDNA Organization of prokaryotic chromosome and plasmids. Chromosome Plasmids 60 Define footer - presentation title / department https://bio1903.nicerweb.com/Locked/media/ch27/DNA.html MUNI SCI Models of DNA replication • Theta • Rolling circle • Linear 61 Department of Experimental Biology MUNI SCI Theta model of DNA replication • Two replication forks can proceed independently around the DNA ring and when viewed from above the structure resembles the Greek letter "theta" (0). • Originally discovered by John Cairns, it led to the understanding that bidirectional DNA replication could take place. • Theta replication is a type of common in E. coli and other organisms possessing circular DNA. Replication fork Origin of -replication Replication bubble Figure 12.4b Genetics: A Conceptual Approach. Fifth Edition © 2014 W. H. Fre«man and Company Department of Experimental Biology https://sacbiotech.files.wordpress.com/2017/11/the-theta-mode-of-dna-replication-in-escherichia-coli.pdf I SCI Theta model of DNA replication Origin of replication □ Double-stranded DNA unwinds at the replication origin,... Q Eventually two circular DNA molecules are produced. • Replication fork Newly synthesized DNA Replication bubble t The forks proceed around the circle. ...producing single-stranded templates for the synthesis of new DNA. A replication bubble forms, usually having a replication fork at each end. Conclusion: The products of theta replication are two circular DNA molecules. 63 Department of Experimental Biology https://sacbiotech.files.wordpress.eom/2017/11/the-theta-mode-of-dna-replication-in-escherichia-coli.pdf MUNI SCI Rolling-circle replication • Rolling circle replication (RCR) is a process which a circular DNA or RNA molecule is replicated in one direction. • RSR is associated with replication of the o genomes of bacteriophages, o plasmids of Gram-positive and Gram-negative bacteria, o archaeal plasmids, o eukaryotic viruses, o the circular RNA genome of viroids. 64 Department of Experimental Biology https://teaching.ncl.ac.uk/bms/wiki/index.php/Rolling_circle_replication MUNI SCI Rolling-circle replication • In this step the linear copies of the original DNA molecule are converted into 3'-OH at the nick is the growing point where DNA synthesis begins. The inner strand is used as a template. • The 3' end grows around the circle giving rise to the name rolling-circle model. Direction of rolling Nucleotides are added to the 3 -OH group, displacing the 5-P-terminated strand. Elongation of the 3' end continues. A nuclease makes a cut yielding a 3 -OH group and a 5'-P group. The 5'-P -terminated strand also is copied. 65 Department of Experimental Biology MUNI SCI Rolling-circle replication (A) One complete revolution Continued DNA synthesis can produce multiple single stranded DNA copies of the original DNA in a continuous Head To Tail series called Concatemer. (B) Two complete revolutions Concatemer 66 Department of Experimental Biology MUNI SCI • Eukaryotic DNA replication MUNI 67 Department of Experimental Biology _ _ T O 0 J. Cell division and reproduction • Every cell needs a complete set of genes, therefore, duplication of the whole genome must occur before cell division. • Each chromosome needs to be accurately duplicated and one copy of the chromosome transferred into each daughter cell. • Estimation, there is around 65 trillion cells (6.5 x 1013) in the body, and there is the same number of replications of the genome. 68 Department of Experimental Biology https://sites.google.eom/a/kcsd.k12.or.us/nickersonl/dna-model-1 MUNI SCI Eukaryotic DNA replication • The basic principles of the eukaryotic DNA replication are the same as in the prokaryotes. Differences: Interphase Mitotic Phase _I_ Mitosis V r- of 2 daughter Cytokinesis ce||s DNA synthesis takes place only at a certain stage of the cell cycle (S-phase) Replication takes place in the nucleus. Multiple replication beginnings- around 10,000 RNA-DNA primers at the Okazaki fragments. At least 15 types of DNA-polymerases. Helicases in on the leading strand. RNAse cleaves the RNA primer. DNA components of chromatin. Interphase Formation Interphase 69 Department of Experimental Biology https://wou.edu/chemisti7/courses/online-chemisti7-textbooks/ch450-and-ch451-biochemistry-defining-life-at-the-molecular-level/chapter-9-dna-replication-and-repair-2/ MUNI SCI Eukaryotic DNA replication Bacteria Eukaryotes Late origin Origin Origin • The origins of replication are present in many copies in the genome (thousands). • The size of the site ori of higher eukaryotes reaches up to several thousand pair base, with proper DNA topology. I • Before the DNA replication is started the RLF (replication licensing factor) binds near the ori I site before replication begins. • RLF coordinates replication initiation from | many ori sites in order to avoid multiple ■ ■= duplication of DNA within one cycle. MUNI 70 Department of Experimental biology Cold Spring Harb Perspect Biol 2013;5:a010108 O 0 J. Eukaryotic DNA replication • Replication licensing factor (RLF) is present in the nucleus before beginning of replication. • Once the replication starts, the RLF is inactivated by degradation or translocation to the cytoplasm to prevent reinitiation of replication. • RLF is loaded on DNA only when the nuclear membrane is disrupted during mitosis. 71 Department of Experimental biology Prior to replication, nucleus contains active licensing factor After replication, licensing factor in nucleus is inactive; licensing factor in cytoplasm cannot enter nucleus Dissolution of nuclear membrane during mitosis allows licensing factor to associ ate with nuclear material Cell division generates daughter nuclei competent to support replication MUNI SCI Eukaryotic DNA replication • In eukaryotes the origin recognition complex (ORC) is highly conserved six-subunit. • ORC recognizes origins of replications and binds them in ATP-dependent manner. • The Cdc6 and Cdt1 proteins are synthesized exclusively in phase G1. • Together they bind to ORC (origin recognition complex) associated with the ori sites. • The MCM2 - 7 helicase is recruited to the origins. • The DNA is unwound. 72 Department of Experimental biology DNA ORC (origin recognition complex) Cdc6' 1 origin I Celt I G, Mem pi f rcplK.it ivo complex (pre-RC) DEGRADATION OF PHOSPHOR Y LATE O WJSV g Cdc6 ■» 4^ — inhibition of Cdtl by geminin PHOSPHORYLATION Of ORC Figure 17-23 Molecular Biology of the Cell Sre (O Garland Science 2008) MUNI SCI Eukaryotic DNA replication • Cdc6 leaves the complex and is phosphorylated and degraded (yeast) or exported from the nucleus due to phosphorylation by CDKs (higher eukaryotes). • Other proteins are attached, which are necessary for binding of DNA-polymerases. • Cdt1, is released from the complex and inhibited by binding of Gemnin. • The cell enters the S-phase. • Since Cdc6 and Cdt1 factors can't be activated again in the same cycle, thus these factors establish DNA replication licensing. 73 Department of Experimental biology DNA ORC (origin recognition complex] Cdc6' 1 origin I Celt I G, Mem pi c replic.it ivo complex (pre-RCl DEGRADATION OF PHOSPHORYLATED 4^ — inhibition of Cdtl by geminin PHOSPHORYLATION OF ORC Figure 17-23 Uolccul.it Biology of the Cell Sit (O Garland Science 20081 MUNI SCI Eukaryotic DNA replication Interphase Mitosis Interphase -i r n r Telomere — Origin of replication Centromere Origin of replication 74 Department of Experimental biology Replicon MUNI SCI Origins of replications Organism Number of replicons Size of replicons Fork movement E. coli S. cerevisiae D. melanogaster X. laevis M. musculus V. faba 1 500 3 500 15 000 25 000 35 000 4600 kb 40 kb 40 kb 200 kb 150 30QfWb 30 000 bp/min 3 600 bp/min 2 600 bp/min 500 bp/min 2 200 bp/min Differences in speed of DNA synthesis. 75 Department of Experimental biology MUNI SCI Eukaryotic DNA polymerases • Pol a - in a stable complex with DNA-primase, synthesis of Okazaki's fragments, 3-5'exonuclease, absence of 5-3'exonuclease activity for the removal of RNA-primers. • Pol y - mitochondrial DNA synthesis/replication. • Pol 5 - synthesis of the leading strand and completion of the synthesis of the lagging strand, high processivity, 3-5'exonuclease. • Pol s - synthesis of the leading strand. 76 Department of Experimental biology MUNI SCI Eukaryotic DNA polymerases Polymerase? Family Catalytic sunuiiU Associated activities Proposed functions Molecular Human gene Chromosomal Y east gened mms . Ii. ■ ' loculianŕ (alius) a (Alpha} B 165 POLÍ Xp22.l-p2U POLÍ (CDC17) Piimase chiflmosomal Triplication, S phase dteekpoinL, DSB repair P (beta) X POLS Spi 1,2 ■ dRP&AP lyase BEE, tingle strand break repair f (gamma) A 140 POLO lSqíí SÍIP1 3'-*5' awnucles5et dRPlya i AATCCC '■ t>) OmdMiti Of lolumou m to Ira Owrtunging 3 »f«J ol Ira (hronniom* lTAY?YTTAao?TTAT3 -.™ ■ AATCCC -■ CAAUCCCAAUC i Replication of ends of chromosomes • The RNA component of Telomerase (451 bases in humans) includes an 11 base template RNA sequence that is used for the synthesis of new telomere repeat DNA. Thus, telomerase acts as a reverse transcriptase (TERT). c) ftymra«4» ol raw lolomc* ON A utng (•KMTMfiM UNA II timpliW I AATCCC CAAUI 3" b; TAOOOTTAOO i AATCCC '■ «> Bynll**1* of raw 1*io*ra** ONA TIT??* CAAUCCCAAUC 3' ■ AAICCC CAAUCCCAAUC T T Ay^l^T T AOQQT T AOOQ T T AOOOT T AO > 1 AATCCC ONA *yntt***rm) fcy ? 'C«ind» Of Utomniil •c*v*y g) N*w M>d of ()>• chrumowMm ihti rvfvlkCM+on '■ TTA???TTA???TTA??TTTAOO'JTTAa 1 J AATCCCAATCCCAATCCC Long*f VflfKI of (tmnwtonit Ajm to Wwwiim Activity oiom P 86 Define footer - presentation title / department • The 3'CAAUC5' sequence on RNA interacts with the 5'GTTAG3' sequence on DNA. • The remaining 3'CCAAUC5' sequence on RNA acts as a template to fill in the 5'GGTTAG3' sequence on DNA. The process repeats. • The alternative strand of DNA gets filled in using DNA as a template. • Complementary strand is synthesized by DNA-polymerase. MUNI SCI Function of Telomerase j parental strand / 3' rGGGGTTGGGGTTGGGGTTG 5'x incomplete, newly synthesized lagging strand TELOMERASE BINDS TELOMERASE EXTENDS 3' END {RNA-templated DNA synthesis) 3' ^ ]TTGGGGTTGGGGTTGGGGTTG JAACCCC 5' direction of telomere synthesis telomerase with bound RNA template 3' COMPLETION OF LAGGING STRAND BY DNA POLYMERASE (DNA-templated DNA synthesis) rGGGGTTGGGGTTGGGGTTGGGGTTGGGGTTG 1AACCCC ^ACCCCAAC 5' 3' "GGGGTTGGGGTTGGGGTTGGGGTTGGGGTTG ]AACCCC j;CCCAACCCCAACCCC <■ 5' DNA polymerase Telomerase enzymatic activity summary. Department of Experimental biology Clear view of telomerase at last (acs.org') https://cen.acs.org/biological-chemistry/genomics/Clear-view-telomerase-last/96/i18 MUNI SCI Telomerase structure remainder of telomerase RNA rest of chromosome newly synthesized telomere DNA 88 Department of Experimental biology telomerase protein "fingers" „ fingers" region of telomerase RNA used as template • The enzyme is composed of protein and RNA. "palm" - active site # HP Im" of telomerase » K"" '' protein thumb' thumb" MUNI SCI Telomeric sequences in various organisms •TTGGGG - T2G4 u Tetrahymena thermophila a Glaucoma chattoni •TTTTGGGG - T4G4 u Euplotes aediculatus a Oxytricha nova •TTTAGGG - T3A1G3 u Arabidopsis thaliana •TGGG - TG3 u Saccharomyces cerevisiae •TTAGGG - T2A1G3 man and mouse, a Trypanosoma brucei 89 Department of Experimental biology Current Opinion in Structural Biology, Volume 25, April 2014, Pages 104-110 Telomeric repeats - control of the cell cycle • In 1984, Blackburn co-discovered telomerase, the enzyme that replenishes the telomere. • For this work, she was awarded the 2009 Nobel Prize in Physiology or Medicine. hWllVli/A Linear rDNA molecules Tetrahymena thermophila y Artificial minichromosomes Chromosome end DNA Yeast cell s \ Yeast cell 90 Department of Experimental biology http://nobelprize.org Elisabeth Blackburn (1948- MUN1 SCI Shelterins A group of proteins covering telomeres at the ends of the chromosome. Shelterin are protein complexes with DNA remodeling activity that acts together with several associated DNA repair factors to change the structure of the telomeric DNA, thereby protecting chromosome ends. (A) Illustration of the shelterin complex loaded on the telomeric DNA. Shelterin components TRF1 and TRF2 are shown as dimers. Importantly, many complexes bind throughout the telomeric repeats. 1TAGGGTTAGG GTTATTAG AATCC C AATC C CA AT C C C, (B) Illustration of the telomere in the t-loop configuration, where the 3' telomeric overhang has invaded the internal repeats, pairing with the complementary C-rich strand. B ZX-TTAG G G - A--AATCCC- 91 Department of Experimental biology Genes 2019, 10, 318; doi:10.3390/genes10040318 MUNI SCI Shelterins Telomerase complex Shelterin complex 92 Department of Experimental biology https://www.mdpi.eom/2072-6694/12/7/1901 MUNI SCI Length of telomerase and aging Most somatic cells do not have telomerase activity (unlike stem or tumor cells) - telomeres are gradually shortened. Human somatic cells grown in culture pass only to a limited extent the number of divisions (50 - 70 generations) - then the division stops, occurs aging and death (replicative cell senescence). Correlates the length of telomeres and the number of cell divisions through which the cell passed, which indicates her old age and nearness of death. v % o r® 93 Department of Experimental biology https://en.wikipedia.org/wiki/Cellular_senescence Hy if lick s limite - molecular clock Leonard Hayflick, 1961: 3 phases of cell growth. Phase 1: Rapid Division. Phase 2: Slow division. Phase 3: Stopping division followed by aging and cell death. Human cells stop after 50 divisions. 94 Define footer - presentation title / department https://historyofyesterday.com/the-biologist-who-gave-every-human-an-expiration-date-of-125-years-37a0f0e8cbad MUNI SCI Chromatin DNA strands in eukaryotic cells arrange themself in higher conformational structures with help of specialized proteins. Interphase DNA: regular winding by nucleosomes. 0.05 fjm 95 Department of Experimental biology Histones Chromosome ££££££££^} Chromatin fiber "Beads on a string" DNA wound on nucleosomes Double helix MUN SCI Structure of nucleosome • The core of nucleosome is formed by octamer of histones: o 2xH2A (2x) o 2xH2B (2x) o 2xH3 (2x) o 2xH4(2x) • Complete nucleosome is formed by attachment of one molecule of histone H1 • Interactions between nucleosomes mediated by H1 and DNA results in formation of compact structures (30 nm fiber). Nucleosome "bead" (8 histone molecules + 146 base pairs of DNA) Protects DNA from nucleases. 96 Department of Experimental biology MUNI SCI Structure of nucleosome 97 Department of Experimental biology Workman and Kingston, Ann. Rev. Biochem. 67: 545 (1998) https://www.ncbi.nlm.nih.gov/books/NBrK26834/ MUNI SCI Levels of chromosome organization Eukaryotic genomic DNA associates with histones into the chromatin. Heterochromatin (highly condensed, transcriptionally inactive) areas. Euchromatin (relaxed and available for binding, transcriptionally active). Each cell exhibits a specific arrangement of heterochromatin and euchromatin. short region of DNA double helix "boads.' on-a- strin g * form of chromatin 30-nm chromatin fiber of packed nucleosomes Section of chromosome in an extended form condensed section of meta phase chromosome entire mtjta phase chromosome 98 Department of Experimental biology MUNI SCI Duplication of nucleosome in replication forks • EM: nucleosomes maintain their structure and distance from each other on both sides of the replication fork. Nucleosomes - $m Replication forks t.- .:•..•.! --v.'./-: cr-'^.-.' '.'•••.is; -"•V..." ! , *,v , ' * \ •....._•> :--.'-.;vvS'{g!,-Ji.'!S-,7 ' 1 |im • Nucleosomes decay and fold quickly to allow proper DNA replication. • Histones are synthesized preferably during the S-phase. MUNI 99 Department of Experimental biology _ _ T O 0 J. Ncleosome replication Old H3/H4 New H3/H4 MCM Helicase 100 Department of Experimental biology Translocation of MCM helicase along the leading strand disrupts parental nucleosome octamers, resulting in the release of H3-H4 and H2A-H2B. Reassembly of nucleosomes behind the replication fork is mediated by chromatin assembly factors (CAFs). Labeling experiments indicate that nucleosome duplication is predominantly conservative. "Old" and "New" histones are assigned to each daughter strand semi-randomly, resulting in equal division of regulatory modifications. MUNI SCI https://www.wikiwand.com/en/S_phase Ncleosome replication • When eukaryotic DNA is replicated, it complexes with histones. This requires the synthesis of histone proteins and the assembly of new nucleosomes. • Transcription of histone genes is initiated near the end of the G1 phase and the translation of histone proteins occurs throughout S phase. • An H3/H4 tetramer is reused in 1 new strand • H2A/H2B is broken down to 2 dimers which are reused arbitrarily. HM ■ M?8 ■ h3 Mm HM »3 M RSON 101 Department of Experimental biology https://slideplayer.com/slide/10450154/ MUNI SCI Ncleosome replication NAP-1 (^AsfT^) old histones: H2A ■ H2B"H3 H4 new histones: H2A ]H2B H3 H4 V „Ml (cam) Participation of specific proteins: Nap-1 (nucleosome assembly protein 1): is responsible for transfer of histones from the site of their synthesis in the cytoplasm to the nucleus. CAF-1 (chromatin assembly factor 1): ensures the transfer of histones to the site of DNA replication, where nucleosomes are assembled, it also binds to PCNA. 102 Department of Experimental biology hftps ://www.yo utu be. com/watch ?v= FYwxJ nGq_8c MUNI SCI THANK YOU FOR YOUR ATTENTION Paramecium Parlor @AmoebaSistens 103 Department of Experimental biology https://amoebasisters.tumblr.com/image/179129709874 MUNI SCI