MUNI SCI Bi4025en Molecular Biology Mgr. Jiří Kohoutek, Ph.D. 1 Department of Experimental Biology Lecture 2 • Nucleic acids: primary, secondary and tertiary structure of nucleic acids, conformation of DNA and RNA, different conformations of DNA and their significance for biological systems, genetic information and genetic code. 2 Department of Experimental Biology MUNI SCI Hunt for the structure of DNA • Watson and Crick restricted themselves to what they saw as chemically and biologically reasonable in terms of DNA structure. • A breakthrough occurred in 1952, when Erwin Chargaff visited Cambridge and inspired Crick with a description of experiments, he had published in 1947. • Chargaff had observed that the proportions of the four nucleotides vary between one DNA sample and the next, but that for particular pairs of nucleotides__, ■- • guanine and cvtosine • adenine and thymine • the two nucleotides are always present in equal proportions. 3 Department of Experimental Biology MUNI SCI Discovery of DNA structure • 1953: James Watson a Francis Crick derived the structure of DNA based on the following data: • Chemical data: Erwin Chargaff principles: • the concentration of thymine and adenine is the same • the concentration of cytosine and guanine is the same. • Physical data: Maurice Wilkins a Rosalind Franklin after exposure of purified DNA molecules to X-rays, there is a characteristic scattering of rays that signal method of arranging DNA components into a helix. 4 Department of Experimental Biology Structure of DNA Levene's tetranucleotide theories dGMP i z dCMP o 0=0.-0 O o " O--0=Q 0 ? 1 -b-0 —fT. T dTMP ° z CJAMP • Features of the proposed DNA structure allow: o encoding genetic information in the form of ordered bases (denial of Levene's tetranucleotide theories) o replication of DNA molecule is based on complementary pairing of bases. group h. «, I ,* d„„ ,"k ,/H HM MUNI 5 Department of Experimental Biology https://sites.google.eom/a/kcsd.k12.or.us/nickersonl/dna-model-1 O U i. Structure of DNA Chemical properties of bases: Spontaneous mutation of DNA bases can also occur by tautomerization: -ex cnol-kelo (automcrism >Nv ^NH, H I o enol / keto o amino / imino nmintvminm lautoinensni • Tautomerization is a net process by which protons are transferred from one site to another by a series of steps in which the solvent is an intermediary. (A) Standard base pairing arrangements Ck H ~t "Vi o -N -N Thymine (keto) Adenine (amino] (B) Anomalous base pairing arrangement o-h- * o- Thymine (enol) ■H N )-N h 6 Department of Experimental Biology Green Chemistry, An Inclusive Approach, 2018, Pages 109-128 Vh---------on —h-m y\ Vi—c >=n / £---------h-n H Cytosine (amino) Guanine (keta) H H u Guanine (keto) Cytosine (imino) Adenine (amino) MUNI SCI Types of Nucleic acids - DNA • DNA forms the genome of prokaryotes, eukaryotes and DNA-viruses. • gDNA-genomic, mtDNA- mitochondrial, cpDNA-chloroplast, pDNA-plasmid, recDNA- recombinant, rDNA-ribosomal, aDNA-ancient. • cDNA (copy DNA, complementary DNA). • dsDNA- double-stranded , ssDNA- single-stranded, cccDNA- covalently closed circular, ocDNA- open circle, NnDNA- linear. • A-DNA, B-DNA, Z-DNA- conformation influenced by sequence and environment. • Special forms of DNA - C-DNA, D-DNA and E-DNA. Department of Experimental Biology June 1981 Plasmid 5(3):371-3 https://microbenotes.com/different-forms-of-dna-b-form-a-form-z-form/ MU SC Types of Nucleic acids - RNA • RNA - forms the genome of RNA-viruses, in cellular organisms it is a component of ribosomes and perform various functions in the transmission and realization of genetic information. • mRNA - mediator, hnRNA - heteronuclear, tRNA - transfer, rRNA -ribosomal, tmRNA - transfer-messenger RNA • snRNA - small-nuclear, snoRNA - small nucleolar, scRNA - small cytoplasmic, gRNA - guide, crRNA - CRISPR RNA • miRNA, siRNA, shRNA, piRNA • ribozyme: an RNA molecule capable of acting as an enzyme • RNA splicing cleavage (or ligation) of RNA and DNA • Ribosome - peptide bond formation • Viral replication 8 Department of Experimental Biology MUNI SCI Nucleotide Nucleotides • phosphatic acid (PHOPHATE) • pentose (SUGAR) o ribose o deoxyribose • organic base (BASE) o purine base adenine guanine o pyrimidine base cytosine thymine uracil 9 Department of Experimental Biology NUCLEOTIDES A nucleotide consists of a nitrogen-containing base, a five-carbon sugar, and one or more phosphate groups. BASE Nil PHOSPHATE Nucleotides are the subunits of the nucleic acids OH OH SUGAR Alberts etal.: Molecular biology of the cell. Garland Sci. 2008, 2015 MUNI SCI Sugar - Pentose in Nucleic acids Sugar - Pentose • ribose in ribonucleic acids (RNA) • deoxyribose in deoxyribonucleic acid (DNA) • the difference is in the presence or absence of hydroxyl groups on 2-carbon SUGARS HOCH2 OH C 5' PENTOSE a five-carbon sugar 11 |i-D-ribose used in ribonucleic acid two kinds are used OH OH I IOC'I l2 OH Each numbered carbon on the sugar of a nucleotide is followed by a prime mark; therefore, one speaks of the "5-prime carbon," etc. [i-D-2-deoxyribose used in deoxyribonucleic acid 10 Department of Experimental Biology Alberts etal.: Molecular biology of the cell. Garland Sci. 2008, 2015 MUNI SCI p-N-glycosidic bond in Nucleic acids A/-glycosidic bond The base is linked to the same carbon (C1) used in sugar-sugar bonds. 11 Department of Experimental Biology Sugar - Pentose • bases are attaching to sugar by (3-N-glycosidic bond which is a nitrogen-carbon linkage between the 1' nitrogen of pyrimidine bases or 9' nitrogen of purines bases and the 1' carbon of the sugar group. N I HC H 2 6 CH CH Pyrimidine Purine https://slideplayer.com/slide/7012073/ Alberts et al.: Molecular biology of the cell. Garland Sci. 2008, 2015 MUNI SCI Pyrimidine and Purine bases in Nucleic acids Bases • pyrimidine (cytosine, thymine, uracil) Bases • purine (adenine, guanine) BASES NH, O HC Nil HC HC, cytosine uracil ,C N I I O H3C "NI i thymine N %o H The bases are nitrogen-containing ring compounds, either pyrimidines or purines. 'N PYRIMIDINE .N N 'N N PURINE NH-, adenine ,NSr/ HC' A \ „c ^ch N' I I N O .C •\JI I # II hc; II G guanine n \ N OH OH J Adenosine 5'-monophosphate (AMP) ^-Y-' Adenosine 5'-diphosphate (ADP) _J -Y- Adenosine S'-triphosphate (ATP) 14 Department of Experimental Biology https://basicmedicalkey.com/nucleotides/ Alberts etal.: Molecular biology of the cell. Garland Sci. 2008, 2015 MUNI SCI Nucleosides and Nucleotides in Nucleic acids • Nucleotides are phosphorylated Nucleosides. NOMENCLATURE BASE NUCLEOSIDE ABBR. adenine adenosine A guanine guanosine G cytosine cytidine C uracil uridine u thymine thymidine T A nucleoside or nucleotide is named according to its nitrogenous base. Single letter abbreviations are used variously as shorthand for (1) the base alone, (2) the nucleoside, or (3) the whole nucleotide— the context will usually make clear which of the three entities is meant. When the context is not sufficient, we will add the terms "base", "nucleoside", "nucleotide", or—as in the examples below—use the full 3-letter nucleotide code. AMP - adenosine monophosphate dAMP = deoxyadenosine monophosphate UDP = uridine diphosphate ATP = adenosine triphosphate BASE + SUGAR = NUCLEOSIDE BASE + SUGAR + PHOSPHATE = NUCLEOTIDE MUNI 15 Department of Experimental Biology Alberts et al.: Molecular biology of the cell. Garland Sci. 2008, 2015 _ _ O 0 J. Nucleosides and Nucleotides in Nucleic acids TABLE 8-1 Nucleotide and Nucleic Acid Nomenclature Base Nucleoside Nucleotide Nucleic add Purines Adenine Adenosine Adenylate UNA Deoxyadenosine Deoxyadenylate DNA Guanine Guanosine Guanylate RNA Deoxyguanosine Deoxyguanylate DNA Pyrimidines Cytosine Cytidine Cytidylate RNA Oeoxycytidine Deoxycytidylate DNA Thymine Thymidine or deoxythymidine Thymidylate or deoxythymidylate DNA Uracil Uridine Uridylate RNA Note: "Nucleoside" and "nucleotide" are generic terms that include both ri bo- and deoxyribo- forms. Also, ribonucleosides and ribonucleotides are here designated simply as nucleosides and nucleotides {e.g., riboadenosine as adenosine), and deoxyribonucleosides and deoxyribonucleotides as deoxynucleosides and deoxy nucleotides (e.g., deoxyriboadenosine as deoxyadenosine). Both forms of naming are acceptable, but the shortened names are more commonly used.Thymine is an exception;"ribothymidine" is used to describe its unusual occurrence in RNA. Table 8-1 Lehninger Principles of Biochemistry, Fifth Edition 0 2008 W. H. Freeman and Company 16 Department of Experimental Biology Lehningeret al.: Principles of Biochemistry, W. H. Freeman and Company, 2008 MUNI SCI Deoxy ribonucleotides Nucleotide is composed of a base (adenine, guanine, cytosine, thymine) attached to a sugar (deoxyribose) to form a nucleoside. The nucleoside with an attached phosphate group makes it nucleotide. The name of the nucleoside containing the base adenine is deoxyadenosine and if the phosphate group is attached at the carbon numbered 5' (five prime) then the formal name of the nucleotide is 2'deoxyadenosine 5'-monophosphate (dAMP). 2'-Deoxyadenosine5'-monophosphate (Deoxyadenylate, dAMP) OH H 2'-Deoxyguano$ine 5'-monophosphate (Deoxyguanylate, dGMP) e 1 uO — P = 0 I 0 1 CH H,C OH H 2'-Deoxycytidine 5'-monophosphate (Deoxycytidylate, dCMP) 2'-Deoxythymidine S'-monophosphate (Thymidylate,dTMP) Figure 19-9 Principles of Biochemistry, 4/e © 2006 Pearson Prentice Hall,Inc. 17 Department of Experimental Biology https://courses.lumenlearning.com/microbiology/chapter/structure-and-function-of-dna/ https://sandwalk.blogspot.com/2007/07/dna-is-polynucleotide.html MUNI SCI Molecule DNA - synthesis Phosphodiester bonds form between the phosphate group attached to the 5' carbon of one nucleotide and the hydroxyl group of the 3' carbon in the next nucleotide. Regular alternation of the sugar-phosphate-sugar-phosphate motif forms the backbone of the polynucleotide: sugar-phosphate (pentose phosphate) backbone. Chains have chemical polarity: 1 end contains phosphate (5-end), the other contains hydroxyl group (3-end). Elongation (synthesis) of polynucleotide chain always runs in the direction 5' - 3'. ? ff) II o i— ester bond phosphodiester -bond 1— ester bond 18 Department of Experimental Biology https://courses.lumenlearning.com/microbiology/chapter/structure-and-function-of-dna/ MUNI SCI Two types of nucleic acid DNA usually double-stranded molecule Strands are bond by hydrogen bonds between base pairs o adenine - thymine o guanine - cytosine RNA usually single-stranded molecule instead of thymine there is uracil. DOUBLE-STRANDED SUGAR,*PHOSPHAT£ DEOXVR180SE O usually Single-strandei sugar* phosphate ought Co. 19 Department of Experimental Biology https://www.thouqhtco.com/dna-versus-rna-608191 M U N I 0 u 1 Differences between DNA and RNA Comparison DNA RNA Full Name Deoxyribonucleic Acid Ribonucleic Acid Function DNA replicates and stores genetic information. It is a blueprint for all genetic information contained within an organism. RNA converts the genetic information contained within DNA to a format used to build proteins, and then moves it to ribosomal protein factories. Structure DNA consists of two strands, arranged in a double helix. These strands are made up of subunits called nucleotides. Each nucleotide contains a phosphate, a 5-carbon sugar molecule and a nitrogenous base. RNA only has one strand, but like DNA, is made up of nucleotides. RNA strands are shorter than DNA strands. RNA sometimes forms a secondary double helix structure, but only intermittently. Length DNA is a much longer polymer than RNA. A chromosome, for example, is a single, long DNA molecule, which would be several centimetres in length when unravelled. RNA molecules are variable in length, but much shorter than long DNA polymers. A large RNA molecule might only be a few thousand base pairs long. Sugar The sugar in DNA is deoxyribose, which contains one less hydroxyl group than RNAs ribose. RNA contains ribose sugar molecules, without the hydroxyl modifications of deoxyribose. 20 Department of Experimental Biology https://www.technologynetworks.com/genomics/lists/what-are-the-key-differences-between-dna-and-rna-296719 MUNI SCI Differences between DNA and RNA Full Name Deoxyribonucleic Acid Ribonucleic Acid Sugar The sugar in DNA is deoxyribose, which contains one less hydroxyl group than RNA's ribose. RNA contains ribose sugar molecules, without the hydroxyl modifications of deoxyribose. Bases The bases in DNA are Adenine (A'), Thymine (T), Guanine ('G') and Cytosine ('C'). RNA shares Adenine (A), Guanine ('G') and Cytosine ('C') with DNA, but contains Uracil ('U') rather than Thymine. Base Pairs Adenine and Thymine pair (A-T) Cytosine and Guanine pair (C-G) Adenine and Uracil pair (A-U) Cytosine and Guanine pair (C-G) Location DNA is found in the nucleus, with a small amount of DNA also present in mitochondria. RNA forms in the nucleolus, and then moves to specialised regions of the cytoplasm depending on the type of RNA formed. Reactivity Due to its deoxyribose sugar, which contains one less oxygen-containing hydroxyl group, DNA is a more stable molecule than RNA, which is useful for a molecule which has the task of keeping genetic information safe. RNA, containing a ribose sugar, is more reactive than DNA and is not stable in alkaline conditions. RNA's larger helical grooves mean it is more easily subject to attack by enzymes. Ultraviolet (UV) Sensitivity DNA is vulnerable to damage by ultraviolet light. RNA is more resistant to damage from UV light than DNA. 21 Department of Experimental Biology https://www.technologynetworks.com/genomics/lists/what-are-the-key-differences-between-dna-and-rna-296719 MUNI SCI Base pairing DNA base pairing Base pairing is due to hydrogen bonds of opposite bases. between two strings = duplex between three strings = triplex between four strings = quadruplex. Hydrogen bond acceptors Hydrogen bond donors O - H-N H N. H-N O Thymine or Uracil 1 donor 1 acceptor N N Adenine 1 donor 1 acceptor N -i H-N^PN N \ O H-N Cytosine 1 donor 2 acceptor \ H Guanine 2 donor 1 acceptor Jack Westin 22 Department of Experimental Biology https://jackwestin.com/resources/mcat-content/nucleic-acid-structure-and-function/base-pairing-specificity-a-with-t-g-with-c MUNI SCI Base pairing- tautomerization (a) Standard base-pairing arrangements H CH3 O—H—N W V H—C N—H—N4 \ / N—C ' V / Thymine (keto) Adenine (amino) \ N—H—O H \ J O—H—N \ H Cytosine (amino) (b) Anomalous base-pairing arrangements Guanine (keto) H—C CH3 \ / C—C O—H -Q H — C 4 V-H-/ H- Thymine (enol) Guanine (keto) Cytosine (imino) Adenine (amino) Standard base-pairing arrangements of the canonical nucleotide isomers. Anomalous base-pairing arrangements of the tautomers. Tautomerization - process when a nonstationary proton tunnels from a common location to a less-common position within the aromatic ring. When tautomerization occurs during replication, the DNA sequence will be "misread", and anomalous base-pairing will occur: such as C* with A, or T*. 23 Department of Experimental Biology https://www.researchgate.net/publication/334974633_Unified_Physics_and_the_Entang lement Nexus of Awareness MUNI SCI Nucleic Acid is a polymer - macromolecules Watson - Crick pairing rules DNA Adenine (amino form) Thymine (keto form) 0-------H —N Guanine (keto form) N H - -0 N H Cytosine (amino form) 25 Department of Experimental Biology MUNI SCI Watson - Crick pairing N—H------O Adenine Uracil (amino form) (keto form) MUNI 26 Department of Experimental Biology Studies in History and Philosophy of Biological and Biomedical Sciences 42 (2011) 119-128 SCI Chargaff's rules qq g Rule 1: (SO " © •The amount of Adenine- equals the amount of Thymine Purim's = • The amount of Guanine ~ equals the amount of Cytosine • The amount of purine = the amount of pyrimidine A + G _ T + A Rule2: TVc = 1 "cTg ^ 1 • The amount of A+T / amount of G+C • This ratio varies among different organisms, but same in different tissues of the same organism. agggggggaagg purines 3 + 9 12 9 + 9 18 tcccccc'cttcc pyrimidines 3 + 9 12 3 + 3 6 is different (%GC) 27 Department of Experimental Biology https://www.biologyexams4u.com/2021/05/chargaffs-experiment-and-why-is.html MUNI SCI Abundance of nucleotides in different organisms • Erwin Chargaff's Data (1950 - 1951) Bos* composition, mole percent Base ratlos Asymmetry ratio A+T A C C T A/T C/C Pu/Py C + C Animals Man Kl'l 19 0 19 8 29.4 1.05 1.00 1 04 1.32 BnMB 29.3 .'I 4 21.0 26.3 1.03 1.02 1.03 1.36 Hen 28.8 20.3 21.S 29.2 1.02 0.95 0.97 1.36 Turtle 29.7 220 21.3 27.9 1.05 1.03 1.00 1.31 Salmon 29.7 206 20 4 29.1 1 02 1 02 1 02 1.43 Si-.i urchtn 32.B 17 7 17.3 32.1 1.02 1.02 1.02 1 56 Uocun 29.3 20 5 20.7 20.3 1.00 1 00 1.00 1.41 Planu Wheat (term 27.3 22.7 22.6 27.1 1.01 1 00 1.00 1.10 Yeast 31.3 18 7 17.1 32.0 005 1 Iľl 1 00 1.79 Aspergillus niger (mold) 25.0 25.1 2S.0 24.9 1 00 1 00 1 00 1.00 Bacteria E.coil 24.7 26.0 25.7 23.6 1 04 1.01 1.03 0.93 Staphylococcus aureus 308 21 0 19 0 29.2 LOS 1.11 1.07 1.50 Clostridium perfringens 38.9 140 12.6 36.3 1.01 1.09 104 2.70 Brucella abortus 21.0 29.0 28.9 21.1 1 00 1 00 1.00 0.72 Sorcina Jufea 13.4 37.1 37.1 12.4 i.oe t 00 1 04 0.35 Bacteriophage* T7 260 24.0 24 0 26.0 1.00 100 1 00 1 08 21.3 26.6 27.2 22.9 0.92 1.05 1.00 o - 1 »1- 1 y i AflliOtfSlI loop SecofldaFV ■ Bass pa>rs (ft) • The base-pairing of complementary nucleotides gives the secondary structure of a nucleic acid. • In a double-stranded DNA or RNA, this refers to the Watson-Crick pairing of complementary strands. • In a single-stranded RNA or DNA, the intramolecular base pairs between complementary base pairs determines the secondary structure of the molecule. MUNI 46 Department of Experimental Biology https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure- replication-and-repair/dna-and-rna-structures ^ Tertiary structure of DNA Acceptor Sierr □ toop variable Anlüüdiüi stsrn Anticodon loop The tertiary structure of a nucleic acid refers to the three-dimensional arrangement of the nucleic acid - that is, the arrangement of the molecule in space, as in the tertiary structure of tRNA. k'l-.lf. r--:c.) i: 47 Department of Experimental Biology https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures MUNI SCI Quaternary structure of DNA Quaternary structure refers to the large shapes and structures that can be made by nucleic acids. DNA - histone = nucleosome RNA - protein = ribosome Yeast H3J 48 Department of Experimental Biology https://www.pngwing.com/en/free-png-ptbih Cell, Vol. 107, 373-386, November 2, 2001 Organization of dsDNA • The DNA double helix may be arranged in space, in a tertiary arrangement of the strands. • The two strands of DNA wind around each other. In a covalently closed circular DNA (cccDNA), this means that the two strands can't be separated. 49 Department of Experimental Biology https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures NucleicAcids Research, 2014, Vol. 42, No. 20, 12404-12414 MUNI SCI Organization of dsDNA Open circular Super colied (OC) dsDNA (SC) dsDNA oc sc 50 Department of Experimental Biology MUNI SCI Organization of dsDNA In closed circular DNA the strands can't be separated, the total number of turns in a given molecule of cccDNA is a constant, called the Linking Number, or Lk. The linking number of a DNA is an integer and has two components, Twist (Tw), or number of helical turns of the DNA. Writhe (Wr), number of times the double helix crosses over on itself (these are the supercoils). • Because Lk is a constant, the relationship can be shown by the equation: Lk = Tw + Wr 1 5 10 15 20 25 xxxxxx?bcx>ooobcxxxxxxxxxxxxxxxxx 20, Close ends to make a c*cle (a) Up- 25. 7W* 25. Wt* 0 Relaxed orcle 51 Department of Experimental Biology https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures MUNI SCI Twist = -1, Writhe = 0. Twist = 0, Writhe = -1. Twist = -2, Writhe = 0. Twist = 0, Writhe = -2 I USD 360° Twist = +1, Writhe = 0. Twist = 0, Writhe = +1. Organization of dsDNA The simplest supercoil (writhe), is the shape a circular DNA assumes to accommodate one too many or one too few helical twists. The two lobes of the figure eight will appear rotated either clockwise or counterclockwise with respect to one another, depending on whether the helix is over or underwound. Twist = +2, Writhe = 0. Twist = 0, Writhe = +2. CP Twist = 0, Writhe = -4. Plectonemic Toroidal 52 Department of Experimental Biology • Twist and writhe are interconvertible. • Extra helical twists are positive and lead to positive supercoiling, while subtractive twisting causes negative supercoiling. https://bioJibretexts.org/Bookshelves/Microbiology/Book%3A_Microbiology_(Boundless) MUNI /7%3A_Microbial_Genetics/7.02%3A_Prokaryotic_Genomes/7.2B%3A_Supercoiling SCI Organization of dsDNA Negative superhehx Lks 23. Tw- 23. Wi~ 0 (Righi-handed) Unwound circle • Normally, this DNA would have a linking number equal to 25, so it is underwound. • The DNA double helical structures have the same value of Lk; however, the DNA can be supercoiled, with the two "underwindings" taken up by the negative supercoils. • This is equivalent to two "turns'-worth" of single-stranded DNA and no supercoils. 53 Department of Experimental Biology https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures MUNI SCI Organization of dsDNA • As a general rule, the DNA of most organisms is negatively supercoiled. Positive superhelix Negative superhelix 54 Department of Experimental Biology https://slideplayer.com/slide/4137430/ https://quizlet.com/203493874/lecture-33-flash-cards/ MUNI SCI Organization of dsDNA • Increasing number of superhelixes of plasmid DNA. 55 Department of Experimental Biology https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures MUNI SCI DNA - topoisomerases • DNA - topoisomerases - alter Lk, the linking number of a DNA, by a bond breaking and rejoining process. • Catalyze the formation of transitional breaks in DNA • Break ("nick") = breaking of the phosphodiester bond between . Cut in one DNA strand is catalyzed by neighboring bases. topoisomerase I. • Cuts in both DNA strands are catalyzed by topoisomerase II = DNA-gyrase. 56 Department of Experimental Biology https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures MUNI SCI DNA - topoisomerase I • Type I topoisomerases (sometimes called "nicking-closing enzymes") carry out the conversion of negatively supercoiled DNA to relaxed DNA in increments of one turn. • Type I , increases Lk by increments of one to a final value of zero. • Type I topoisomerases are energy independent, because they don't require ATP for their reactions. • Anti-tumor drugs, including Camptothecin, target the eukaryotic topoisomerase I. 57 Department of Experimental Biology t *or*c *ou*oubo*» DNA M rumúit otoůt vtftodtn k dnMinu bono jixnii:: rwMopoooRiMa i M kovilifltni pApopft klo«l*i»DKA,imlpW. I ONA Ml flyfti fliOhOU tostorJeÄTOv* wby 'mmSbtn P OH " —P—p-- **'-*p4. tiilodiima) it iiiDi uotrovu|tjat tfiCutroubcwe» ON*. v puvodf* tonne HO •0 : nun:: MUNI SCI DNA - topoisomerase I • Releases superhelix tension from the superhelix DNA. • Topoisomerase covalently attaches to one of the phosphates in DNA, o cuts DNA strand can rotate around its longitudinal axis, o the strand tension/pressure is relieved, o double helix restoration and enzyme is released. Double stranded helix v Double helix (-1 Binding of Topo I Topo I \ Nick DNA; form covalent DNA-phosphotyrosine bond under other strand and reseaf DNA Pass cut 3' end Dissociation of TopoI 3 negative supercoils 2 negative supercoils 58 Department of Experimental Biology https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures MUNI SCI DNA - topoisomerase II (DNA Gyrase) • Type II topoisomerases (sometimes called DNA gyrases) reduce Lk by increments of two. • These enzymes are ATP-dependent and will alter the linking number of any closed circular DNA. • TOP2 functions by relieving supercoils generated as a function of the double stranded nature of DNA. • Type II topoisomerases act on naturally occurring DNAs to make them supercoiled. Supercoiled Relaxed, decatenated Catenated 59 Department of Experimental Biology https://www.cliffsnotes.com/study-guides/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-and-rna-structures MUNI SCI DNA - topoisomerase II (DNA Gyrase) • Type II topoisomerases mechanism of action. • The enzyme unknots and untangles DNA by passing an intact helix through a transient double-stranded break that it generates in a separate helix. 60 Department of Experimental Biology Front. Neurol. 9:459. doi: 10.3389/fneur.2018.00459 MUNI SCI Differences between topoisomerases I and II Supercoiled DNA Type I Type II -> 61 Department of Experimental Biology Nick in one strand Nick in both strands • the one strand moves above the other one in the cut and merges: • L is reduced by 1 both strands move above the break and merges: + ATP for function • L is reduced by 2 MUNI https://www.sigmaaldrich.com/CZ/en/technical-documents/protocol/genomics/sequencing/sanger- C P sequencing Differences between topoisomerases I and II 62 Define footer - presentation title / department MUNI SCI DNA organization in regulation of trascription Eukaryotic DNA is linear and double-stranded. • It binds to protein scaffolding. • Generates superhelixes, solenoid loops and relaxed areas. negative superceding left-handed mitleosomc 0« podive tu pef coiling right-handed nucleoid me topologies I ly associating domain (TAD) transcription factories (condensates) / "I supercoiled DNA promoter enhancer cohesin complex CTCF DNA topoisomerase II 63 Department of Experimental Biology Nanoscale. 2017 Feb 2; 9(5): 1862-1870. Nucleic Acids Research April 2020 48(9):4614-4626 MUNI SCI DNA sequences adopting alternative structures • Unique DNA sequence: ....AATGCTGATGTCTGACTCGGA... • Repetitive sequences or repeat. • Terms: unit of repetition, length of unit of repetition, frequency of repetition. • Example: ATG... ATG....ATG....ATG...unit = ATG, length = 3 nucleotides, frequency = 4x. • Tandem repeats - tied tightly to each other "head to toe".. ATGCATGCATGC • Direct repetition (5....ATGC.....ATGC.....3) repeats on the same strings in the same direction (5' 3). MUNI 64 Department of Experimental Biology Dig Dis Sci. 2015 Aug; 60(8): 2230-2231. p> o t DNA sequences adopting alternative structures Inverted repetition: repeated on the second string in the reverse direction -potential for creating a hairpin or hairpin with a loop. 5'...ATGCGCAT...3' palindrome (hairpin/hairpin with loop) 3'...TACGCGTA...5' 5 ... ATGCXXXXXGCAT... 3' 3'... TACGYYYYYCGTA... 5' hairpin with loops (within dsDNAthe cross structure is established) LTR - long terminal repeat 5'-ATGC...GCAT............ 3'-TACG...CGTA............ Palindrome T T A G C A C G T G C T A A 1 I I I I I T ATGC...GCAT-3' TACG...CGTA-5' J_I I I_l_l_L AATCGTGCACGATT Mirror repeat TTAGCACCACGATT ._l_l i l 1 l l l_...... AATCGTGGTGCTAA 65 Department of Experimental Biology MUNI SCI Sheme of hairpin structure eI ._TGCGAT ACTC ATCGC A_. 5 4 TTT I I I I TTI * 3 5'« TTT TTT Hairpin * 3' 5'«- 3'«- _TGCGATACTCATCGCA TTT I I I I '''' nr J_LL ACGCTATCAGTAGCGT 3*4 Cruciform -► 3' 5' 66 Department of Experimental Biology MUNI SCI Denaturation and Renaturation of DNA and RNA Denaturation of DNA DNA is a highly stable molecule. Non-covalent hydrogen bonds between the 2 complementary strands can be broken. The DNA strands can be separated. This is called denaturation. 'XIÜOOflKMK 5 5' 3' DNA is denatured by heating c AG G CCGAGTGTCC llllLUliii/Zu^ 67 Department of Experimental Biology https://elearning.fondation-merieux.org/molecular-biology/chapter-1/page-15.php MUNI SCI Denaturation and Renaturation of DNA and RNA • Denaturation of dsDNA = transformation of dsDNA into ssDNA. • Renaturation of ssDNA = transformation of ssDNA into dsDNA. • Induction of denaturation: o by increasing the temperature of the solution, o by changing the pH from neutral to alkaline or acidic. • Occurs in vitro and naturally also in vivo. Native double Strands Separated Double helix helix unwinding strands reformed ■ FIGURE 9.1 9 Helix tin winding in DNA denaturation. The double helix unwinds when DNA is denauired, wiih eveiuual separation oFuhe strands. The double helix is re-formed on rena-tumtion ■willi slow cooling and annealing. 68 Department of Experimental Biology https://www.brainkart.com/article/Denaturation-of-DNA_27539/ MUNI SCI Denaturation and Renaturation of DNA and RNA dsDNA denaturation is manifested by hyperchromic effect, which means increased absorbance of UV-light with a wavelength 260 nm. Value Tm or melting point = temperature, in which 50% of dsDNA molecules are denatured. Tm depends on the content of the bases. Absorbance at 260 nm single stranded DNA VVV AAA DNA partially unwind Double / stranded / DNA / T = 69°C m 30 40 50 60 70 80 Temperature (°C) r 90 100 69 Department of Experimental Biology https://elearning.fondation-merieux.org/molecular-biology/chapter-1/page-15.php MUNI SCI Denaturation curve Other options for determining %GC: • Ultracentrifugation in CsCI • HPLC. 100 Tm= 69,3 * 0,41 (CC). GC = _ Tm - 69,3 0,41 70 Department of Experimental Biology 50 Poly (AT) Naturally occurring DNA with A/Tand G/C base pairs —r-1-1 i 10 30 50 PolylGQ Temperature (CC) • GC = molar fraction of guanine and cytosine in DNA, • 69.3 and 0.41 are empirically the coefficients laid down, pro poly(AT) Tm = 69,3. https://sandwalk.blogspot.com/2007/12/dna-denaturation-and-renaturation-and.html MUNI SCI Hybridization of DNA and RNA • Hybridization is the process of combining two complementary single-stranded DNA or RNA molecules and allowing them to form a single double-stranded molecule through base pairing. • The more hybridizing molecules coincide in sequences, or the higher their sequential homology, the greater it is the probability of their hybridization. Double Stranded Single Stranded Double Stranded 71 Department of Experimental Biology DNA-Based Storage: Trends and Methods, July 2015, DOI: 10.1109/TMBMC.2016.2537305 MUNI SCI Hybridization of DNA and RNA Use for evaluation of the degree of sequential/structural similarity of DNA without sequencing. Prepare DNA Separate strands and Cool and allow to re-anneal mix Sample 1 Sample 2 MXMM> Sample 3 72 Department of Experimental Biology High melting temperature = Samples 1 and 2 are closely related Low melting temperature = Samples 1 and 3 are not closely related MUNI SCI Fly T -G T A G A ^ C A T C - G C-l ■C G A C A C T GH T G C -he G H C GJ Hybridization of DNA and RNA Human ■-T A -i G G A T A T G C - H A - C G - A T - -C G - A T T A T A KG CH Chimp T G A A. G A C A C A T G A -i C T T C-T Q-T T 4. QH T A cH G C -C G ■■ Double-helical DNA from two species is unwound (denatured), cut, and mixed. Complementary pieces bind (called reannealing), forming some hybrid DNAs—one strand from each species. Then the temperature is raised and the rate of interspecies double-helix separation determined. The higher the temperature needed to separate the hybrid DNA, the more similar the DNA sequences must be. Fly/human HurňarVctnimp (leas cíHiservaiion) (more conservation) 73 Department of Experimental Biology https://www.sciencedirect.com/topics/medicine-and-dentistry/dna-hybridization MUNI SCI Usage of Hybridization in the reasearch Identification of specific DNA a RNA sequences. Estimation of their structural similarity. PCR. Transcription in vitro. FISH. DNA hybridization single-stranded DNA probes for gene A hybridization in 50% formamide at 42°C only A forms stable double helix ieA / mixture of single-stranded DNA molecules JL hybridization in 50% formamide at 35°C imperfect J base-pairing A A, C, and E all form stable double helices 74 Department of Experimental Biology Figure 8-25. Molecular Biology of the Cell, 4th Edition. MUNI SCI T Usage of Hybridization in the reasearch SCI Codex System Antibody Barcode lllllllllllll Reporters II HI lllllllllll I II + "I LULL Barcode Reporters lllllllllll I II lllllllllll I II UNI 75 Define footer - presentation title / department Akoya Biosystems THANK YOU FOR YOUR ATTENTION IF I AM LOST IN TRANSLATION 76 Department of Experimental Biology base {here: guanine} q n _>NH :ibose O OH phosphaie Q=P—0^ Ô" JUST BLAME MY RNA https://www.jokejive.com/topic/molecular+biology#&gid=1&pid=8 MUNI SCI