DNA Structure Chapters 10&11 Biochemistry by Reginald Garrett and Charles Grisham

Garrett and Grisham, Biochemistry, Third Edition

DNA Structure Chapters 10&11

Biochemistryby

Reginald Garrett and Charles Grisham

Igor ChesnokovDepartment of Biochemistry and Molecular GeneticsOffice Phone # 934-6974E-mail: [email protected]


Genome sizes compared.Human genome contains a 1000 times asmany nucleotide pairs as the genome of typical bacteria, 20 times as many genes,and about 10,000 times as much non-coding DNA (~98.5% of the genome for a human is non-coding, as opposed to ~11% of thegenome for E. coli).

The amount of DNA content doesn’t reflect ourestimation of the “complexity” of the organism.


Central Dogma of Molecular Biology Information Transfer in Cells

• Information encoded in a DNA molecule is transcribed via synthesis of an RNA molecule

• The sequence of the RNA molecule is "read" and is translated into the sequence of amino acids in a protein.


DNA is the repository of genetic information (replication), RNA serves in the expression of this information through the processes of transcription and translation.


Essential Questions

• What are the structures of the nucleotides? • How are nucleotides joined together to form

nucleic acids? • What is the higher-order structure of DNA• What are the biological functions of

nucleotides and nucleic acids?


DNA structure 1• What Is the Structure and Chemistry of

Nitrogenous Bases?• What Are Nucleosides? • What Is the Structure and Chemistry of

Nucleotides?• What Are Nucleic Acids? • What Are the Different Classes of Nucleic

Acids?


DNA structure 2

• What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt?

• Can the Secondary Structure of DNA Be Denatured and Renatured?

• What is the Tertiary Structure of DNA?• What Is the Structure of Eukaryotic

Chromosomes?


Structure and Chemistry of Nitrogenous Bases

The bases of nucleotides and nucleic acids are derivatives of either pyrimidine

or purine. • Pyrimidines

– Cytosine (DNA, RNA)– Uracil (RNA)– Thymine (DNA)

• Purines – Adenine (DNA, RNA)– Guanine (DNA, RNA)


The pyrimidine ring system (six-membered heterocylic aromatic ring with two nitrogen atoms); by convention, atoms are numbered as indicated. (b) The purine ring system consists of two rings (pyrimidine and imidazole), nine atoms numbered as shown.

Both are relatively insoluble in water due to aromatic character.


The common pyrimidine bases – cytosine (DNA and RNA), uracil (RNA), and thymine (DNA).


The common purine bases —adenine and guanine (found in both DNA and RNA).


What Are Nucleosides?When a base is linked to a sugar it forms a Nucleoside.

• The sugars are Pentoses• D-ribose (in RNA) • 2-deoxy-D-ribose (in DNA) • The difference - 2'-OH vs 2'-H • This difference affects secondary structure and stability of

nucleic acids• Base in nucleosides is linked to a sugar via a glycosidic

bond • 1’C of sugar links to 9 N of purine or to the 1 N of pyrimidine

base• Sugars make nucleosides more water-soluble than free

bases


Pentose in a five membered ring is known as furanose. Furanose structures (ribose and deoxyribose) are presented above. Presence of a hydroxyl group at the 2-position has dramatic effect on secondary structures available to DNA and RNA as well as their susceptibilities to hydrolysis. DNA is more stable.


b-Glycosidic bonds link nitrogenous bases and sugars to form nucleosides.

1’C links to 9 N of purine and to the 1 N of pyrimidine base.


The common ribonucleosides—cytidine, uridine, adenosine, and guanosine. Also, inosine (uncommon nucleoside) is drawn.


Example of syn and anti conformation.

Anti conformation is adopted in pyrimidine nucleosides (syn conformation is sterically hindered) and favored in purine nucleosides.


Structure and Chemistry of Nucleotides

Nucleotides or Nucleoside phosphates result when phosphoric acid is esterified to a sugar-OH group of nucleoside (at C-5)• Most nucleotides are ribonucleotides • Nucleotides have acidic properties• Nucleic acids, which are polymers of

nucleosides derive their names from the acidity of phosphate groups.


Structures of the four common ribonucleotides —AMP, GMP, CMP, and UMP—together with their two sets of full names, for example, adenosine 5'-monophosphate and adenylic acid. Also shown is the nucleoside 3'-AMP (uncommon, product of hydrolysis).


Functions of Nucleotides• Facts to remember: • NTPs and dNTPs are substrates for nucleic acids• Bases serve as recognition units or information symbol

but not involved in the biochemistry of metabolism• ATP is central to energy metabolism (energy currency) • GTP drives protein synthesis • CTP drives lipid synthesis • UTP drives carbohydrate metabolism• Cyclic nucleotides are signal molecules and regulators

of cellular metabolism and reproduction• Nucleoside 5'-triphosphates are carriers of energy.

Energy is stored in phosphoric bonds.


Phosphoryl and pyrophosphoryl group transfer, the major biochemical reactions of nucleotides. Phosphoric bonds are prime source of chemical energy to do biological work (ATP, GTP, CTP and UTP, also deoxy- counterparts).


Cyclic nucleotides are cyclic phosphodiesters.

Structures of the cyclic nucleotides cAMP and cGMP. Phosphoric acid is esterified to two of the available ribose hydroxyl groups.

Important! They are regulators of cellular metabolism and are found in all cells.


What are Nucleic Acids? Polynucleotides!

• Facts to remember:• Linear polymers of nucleotides linked 3‘C to 5‘C

by phosphodiester bridges • Ribonucleic acid and Deoxyribonucleic acid • Know the shorthand notations • Sequence is always read 5' to 3' • In terms of genetic information, this

corresponds to "N to C" in proteins


3'-5' phosphodiester bridges link nucleotides together to form polynucleotide chains.


Shorthand notations for polynucleotide structures.

Furanoses are represented by vertical lines; phosphodiesters are represented by diagonal slashes in this shorthand notation for nucleic acid structures. Bases serve as distinctive side chains and give the polymer it’s unique identity.


What Are the Different Classes of Nucleic Acids?

• DNA - one type, one purpose • RNA - 3 (or 4) types, 3 (or 4) purposes

– ribosomal RNA - the basis of structure and function of ribosomes

– messenger RNA - carries the message– transfer RNA - carries the amino acids– Small nuclear RNA– Small non-coding RNAs


The DNA Double Helix

• Erwin Chargaff had the pairing data. Chargaff rule – the number of purine residues equals the number of pyrimidine residues in all organisms (A=T, G=C).

• Rosalind Franklin's X-ray fiber diffraction data was crucial (Helix!)

• Watson-Crick model of the DNA double helix.• "Base pairs" arise from hydrogen bonds

Garrett and Grisham, Biochemistry, Third Edition Table 10-3, p.320


The Base Pairs Postulated by Watson

The Watson-Crick base pairs A:T and G:C.


The Base Pairs Postulated by Watson

The Watson-Crick base pairs A:T and G:C.


A model of DNA double helix.

The nucleotides are linked covalently by phoshodiester bonds through the3’-hydroxil (-OH) group of one sugar and the 5’-phosphate (P) of the next. Two DNA strands are held togetherby hydrogen bonds between the paired bases. Two hydrogen bonds form between A and T, while three form between G and C. The bases can pair in this way only if the two polynucleotide chains that contain them are antiparallel to each other.

The coiling of the two strands around each other creates two groves in the double helix.

Consequences – each strand of DNA contains a sequence of nucleotides that are exactly complementary tothe sequence of its partner strand.


Structure of DNAsummary

The fundamental structure of DNA is a Double Helix stabilized by hydrogen bonds!

• DNA consists of two polynucleotide strands wound together to form DNA double helix

• Strands run in opposite direction (antiparallel)• Two strands are held together through inter-

chain hydrogen bonds• These H bonds pair the bases of nucleotides in

one chain to complementary bases in the other – base pairing.


Comparison of A, B, Z DNAABZs of DNA Secondary Structure

• A: right-handed, short and broad, 2.3 Å, 11 bp per turn (dehydrated DNA, probably does not exist in vivo)

• B: right-handed, longer, thinner, 3.32 Å, 10 bp per turn, (most common)

• Z: left-handed, longest, thinnest, 3.8 Å, 12 bp per turn (G-C rich regions)


Comparison of the A-, B-, and Z-forms of the DNA double helix. The distance required to complete one helical turn is shorter in A-DNA than it is in B-DNA. The alternating pyrimidine–purine sequence of Z-DNA is the key to the “left-handedness” of this helix.


How Do Scientist Determine the Primary Structure of Nucleic Acids?

Sequencing Nucleic Acids • Chain termination method (dideoxy method),

developed by F. Sanger • Base-specific chemical cleavage, developed

by Maxam and Gilbert • Both use autoradiography - X-ray film

develops in response to presence of radioactive isotopes in nucleic acid molecules


Chain termination method is based on biochemistry of DNA replication. DNA polymerase copies ssDNA in vitro in the presence of the four deoxynucleotide monomers.

A double-stranded region of DNA must be artificially generated by adding a primer.

DNA polymerases add nucleotides in 5'-3' direction


Chain Termination MethodBased on DNA polymerase reaction

• Run four separate reactions • Each reaction mixture contains dATP,

dGTP, dCTP and dTTP, one of which is P-32-labelled

• Each reaction also contains a small amount of one dideoxynucleotide: either ddATP, ddGTP, ddCTP or ddTTP


The chain termination or dideoxy method of DNA sequencing.

Four reaction mixtures with DNA polymerase, nucleoside triphosphates plus small amounts of the four dideoxynucleotide analogs of these substrates, each of which contains a distinctive fluorescent tag, illustrated here as: Orange for ddATPBlue for ddCTPGreen for ddGTPRed for ddTTP


Chemical Cleavage MethodNot used as frequently as Sanger's

• Start with ssDNA labelled with P-32 at one end • Strand is cleaved by chemical reagents • Assumption is that strands of all possible

lengths will be produced, each cleaved at just one of the occurrences of a given base.

• Fragments are electrophoresed and sequence is read


A photograph of the autoradiogram from an actual sequencing gel. A portion of the DNA sequence of nit-6, the Neurospora gene encoding the enzyme nitrite reductase.


Can the Secondary Structure of DNA Be Denatured and Renatured?

Important for Study of Genome complexity• When DNA is heated to 80+ degrees Celsius,

its UV absorbance increases by 30-40% • This hyperchromic shift reflects the unwinding of

the DNA double helix • Stacked base pairs in native DNA absorb less

light • When temperature is lowered, the absorbance

drops, reflecting the re-establishment of stacking


Steps in the thermal denaturation and renaturation of DNA. Renaturation (re-annealing) depends on DNA concentration and time. The nucleation phase of the reaction is depending on sequence alignment of the two strands. This process takes place slowly because it takes time for complementary sequences to encounter one another in solution and then align themselves in register. Once the sequences are aligned, the strands zipper up quickly.


These c0t curves show the rates of re-association of denatured DNA from various sources and illustrate how the rate of re-association is inversely proportional to genome complexity. The DNA sources are as follows: poly A+poly U, a synthetic DNA duplex of poly A and poly U polynucleotide chains; mouse satellite DNA, a fraction of mouse DNA in which the same sequence is repeated many thousands of times; MS-2 dsRNA, the double-stranded form of RNA of MS-2, a simple bacteriophage; T4 DNA, the

DNA of a more complex bacteriophage; E. coli DNA, bacterial DNA; calf DNA (nonrepetitive fraction), mammalian DNA (calf) from which the highly repetitive DNA fraction (satellite DNA) has been removed. Arrows indicate the genome size (in bp) of the various DNAs.


Tertiary Structure of DNA

• In duplex DNA, ten bp per turn of helix • Circular DNA sometimes has more or less

than 10 bp per turn - a supercoiled state, underwound (-) or overwound (+).

• Enzymes called topoisomerases or gyrases can introduce or remove supercoils


There are toroidal and interwound varieties of DNA supercoiling. (a) The DNA is coiled in a spiral fashion about an imaginary toroid. (b) The DNA interwinds and wraps about itself. (c) Supercoils in long, linear DNA arranged into loops whose ends are restrained—a model for chromosomal DNA.


A simple model for the action of bacterial DNA gyrase (topoisomerase II).

The A-subunits cut the DNA duplex (1) and then hold onto the cut ends (2). Conformational changes occur in the enzyme that allow a continuous region of the DNA duplex to pass between the cut ends and into an internal cavity of the protein. The cut ends are then re-ligated (3), and the intact DNA duplex is released from the enzyme. The released intact circularDNA now contains two negative supercoils as a consequence of DNA gyrase action (4).


Supercoiled DNA in a toroidal form wraps readily around protein “spools.” A twisted segment of linear DNA with two negative supercoils (a) can collapse into a toroidal conformation if its ends are brought closer together (b) Wrapping the DNA toroid around a protein “spool” stabilizes this conformation of supercoiled DNA (c)


A diagram of the histone octamer.

Nucleosomes consist of two turns of DNA supercoiled about a histone “core” octamer.


Nucleosomes as seen in the electron microscope.“Beads on a string” – partially unfolded chromatin.


The 2-nm DNA helix is wound twice around histone octamers to form 10-nm nucleosomes.

These nucleosomes are then wound in solenoid fashion with six nucleosomes per turn to form a 30-nm filament.

The 30-nm filament forms long DNA loops, each containing about 60,000 bp, which are attached at their base to the nuclear matrix.

Eighteen of these loops form a miniband unit of a chromosome.

Approximately 106 of these minibands occur in each chromatid of human chromosome 4 at mitosis.


Structure of Eukaryotic Chromosomes

• Human DNA’s total length is ~2 meters!• This must be packaged into a nucleus that

is about 5 micrometers in diameter• This represents a compression of more

than 100,000!• It is made possible by wrapping the DNA

around protein spools called nucleosomes and then packing these in helical filaments


Each DNA molecule that forms a linear chromosome must contain a Centromere, two Telomeres and Replication Origins

Sequence of events a typical chromosome follows during the cell cycle:The DNA replicates in interphase beginning at the origins of replication and proceeding bidirectionally from theorigins across the chromosome. Many origins are required to ensure that the entire chromosome can be replicated rapidly. In M phase, the centromere attaches the duplicated chromosome to the mitotic spindleso that one copy is distributed to each daughter cell during mitosis. The centromere also helps to hold theduplicated chromosomes together until they are ready to be moved apart. The telomeres form special capsat each chromosome end.

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DNA Structure Chapters 10&11 Biochemistry by Reginald Garrett and Charles Grisham

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