Lec 3. DNA structure, function and metabolism

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DNA structure, function andmetabolism



I. Bacterial Transformation is Mediated byDNA

Exp eriment by Frederick Griffith 1928± Demonstrated first evidence that genes are molecules± Two different strains of Streptococcus pneumoniae

Non- p athogenic = Avirulent = ROUGH cells (R)Pathogenic = virulent = SMOOTH (S)

± Smooth outer covering = ca p sule± Cap sule = slimy, p olysaccharide± Enca p sulated strains esca p e p hagocytosis



± The ca p sule alone did not cause p neumoniaHeat-killed S strain was avirulentAbility to esca p e immune detection and multi p ly

± When heat-killed S strain was mi xed with living R strainthe mouse dies of p neumoniae

Enca p sulated strain (S) recovedred from dead mouseNow a live strainThe R strain had somehow acquired the ability top roduce the p olysaccharide ca p sule

± Transformation± Ability to p roduce coat was an inherited trait

Daughter cells also p roduced ca p sule



Transformation± Up take of genetic material from an e xternal

source resulting in the acquisition of new traits(p henoty p e is changed)

± Griffith s exp riment was the earliest documentevidence of transformation



Avery, MacLeod and McCarty defined the transforming agentof Griffith s e xp eriment as DNA (1944)

± Chemical com p onents of heat-killed S strain bacteria werep urified and co-injected with live R strain

Polysaccharide/CarbohydrateLip idsProteinNucleic acids

± DNA± RNA



II. Viral DNA is Transferred into Cells DuringInfection The Hershey-Chase Exp eriment (1952)

T2 Bacterio p hage studies± Bacterio p hage = viruses that infect bacteria± Major chemical com p onents = DNA and p rotein± E scherichia coli infected with T2 p roduce

thousands of new viruses in the host cellHost cell lyses and p hage are released



Determination of whether DNA or p rotein was directingsynthesis of new p hage p articles

± Viral p roteins were radioactively labeled with:35S by growing T2-infected bacteria in 35S-methionine =1st Batch

± Amino acid labeling± DNA does not contain any sulfur atoms

32P by growing T2-infected bacteria in 32-P± Nucleic acid labeling± Amino acids do not contain p hos p horous



± Radioactively labeled viruses were isolated fromthe culture and used to R EINFECT new host cells

Batch 1 = p rotein labeled

Batch 2 = DNA labeled± Blender used to disru p t p hage on surface of

bacteria from cells and their cyto p lasmiccom p onents then centrifuged

Sup ernatant?? (Protein never entered the cell)Pellet?? (DNA injected into the cell)



Figure 1: Nucleotides have three com p onents .

A nucleotide consists of a p hos p hate grou p , a p entose sugar (ribose or deo xyribose), and a nitrogen-containing base, all linked together by covalent bonds . The nitrogenous bases have two different

chemical forms: p urines have two fused rings, and the smaller p yrimidines have a single ring .

Laying the Groundwork:Levene Investigates the

Structure of DNA



whether there were any differences in DNA among different s pp . develo p ed a new p ap er chromatogra p hy method for se p arating andidentifying small amounts of organic materialChargaff reached two major conclusions (1950) . First, nucleotide com p osition of DNA varies among s p ecies (thesame nucleotides do not re p eat in the same order, as p rop osed byLevene) .

Second, almost all DNA-no matter what organism or tissue ty p e itcomes from-maintains certain p rop erties, even as its com p ositionvaries .

± the amount of adenine (A) is usually similar to the amount of thymine(T)

± the amount of guanine (G) usually a pp roximates the amount of cytosine (C) .

± In other words, the total amount of p urines (A + G) and the totalamount of pyrimidines (C + T) are usually nearly equal .

± (This second major conclusionis known as "Chargaff's rule . )

No idea about A-T and G-C combination

Strengthening the Foundation: Chargaff Formulates His "Rules"



Rosalind Franklin s data p rovide clues aboutDNA s 3-D shap e

X-ray Crystallogra p hy defined± Diffracted X-rays as they

p ass through a crystallizedsubstance

± Patterns of s p ots are translated by mathematicalequations to define 3-D sha p e

± Helix± Width = 2 nm p robably two strands (DOUBL E HELIX)± Nitrogenous bases = 0 .34 nM a p art± One turn every 3 .4 nM (10 base p airs p er turn)



Chargaff's realization that A = T and C = Gcrucially im p ortant X-ray crystallogra p hy work by English researchers RosalindFranklin and Maurice Wilkins ,contributed to Watson and Crick's derivation of the three-dimensional, double-helical model for the structure of DNA . Watson and Crick's discovery was also made

possible by recent advances in model

buildingthe assembly of p ossible three-dimensional structures based u p on knownmolecular distances and bond angles , a technique advanced by Americanbiochemist Linus PaulingUsing cardboard cutouts re p resenting the individual chemical com p onents of thefour bases and other nucleotide subunits, Watson and Crick shifted moleculesaround on their deskto p s, as though p utting together a p uzzle . They were misled for a while by an erroneous understanding of how the differentelements in thymine and guanine (sp ecifically, the carbon, nitrogen, hydrogen, andoxygen rings) were configured . Only u p on the suggestion of American scientist Jerry Donohue did Watson decideto make new cardboard cutouts of the two bases,

P utting the Evidence Together: Watson and CrickP ropose the Double Helix



Figure 3: DNA is a double heli x.

(A) Francis Crick (left) and James Watson (right) p rop osed that the DNA molecule has a double-helical structure .

(B) Biochemists can now p inp oint the p osition of every atom in a DNA molecule .



Although scientists have made some minor changes to the Watson andCrick model, or have elaborated u p on it, since its ince p tion in 1953, themodel's four major features remain the same yet today . These features areas follows:DNA is a double-stranded heli x, with the two strands connected byhydrogen bonds . A bases are always p aired with Ts, and Cs are alwayspaired with Gs, which is consistent with and accounts for Chargaff's rule .

Most DNA double helices are right-handed ; that is, if you were to holdyour right hand out, with your thumb p ointed u p and your fingers curledaround your thumb, your thumb would re p resent the a xis of the heli x and

your fingers would re p resent the sugar- phos phate backbone . Only onetyp e of DNA, called Z-DNA, is left-handed .

The DNA double heli x is anti- p arallel, which means that the 5' end of onestrand is paired with the 3' end of its com p lementary strand (and viceversa) . Nucleotides are linked to each other by their p hos p hate grou ps,which bind the 3' end of one sugar to the 5' end of the ne xt sugar .

Not only are the DNA base p airs connected via hydrogen bonding, but theouter edges of the nitrogen-containing bases are e xp osed and available forpotential hydrogen bonding as well . These hydrogen bonds p rovide easyaccess to the DNA for other molecules, including the p roteins that p layvital roles in the re p lication and e xp ression of DNA



Figure 4 : Base

p airing in DNA iscom p lementary .

The p urines (A andG) p air with thep yrimidines (T andC, res p ectively) to

form equal-sizedbase p airsresembling rungson a ladder (thesugar- p hos p hatebackbones) . The

ladder twists into adouble-helicalstructure .



Nature 171: 737-738 April 1953

Watson JD and Crick FC (1953) M olecular Structureof Nucleic Acids: A Structure for Deoxyribose NucleicAcid .

1962 Nobel Prize awarded to three men Watson,Crick and Wilkins



Other scientists have elaborated on Watson and Crick's model -identification of three different conformations of the DNA double heli x. The most common conformation in most living cells (which is the onede p icted in most diagrams of the double heli x, and the one p rop osed byWatson and Crick) is known as B-DNA . A-DNA, a shorter and wider form that has been found in dehydratedsam p les of DNA and rarely under normal p hysiological circumstances;Z-DNA, a left-handed confirmation . Z-DNA is a transient form of DNA, onlyoccasionally e xisting in res p onse to certain ty p esof biological activity .

Z-DNA was first discovered in 1979, but itsexistence was largely ignored until recently . Scientists have since discovered that certainp roteins bind very strongly to Z-DNA, suggesting thatZ-DNAp lays an im p ortant biological role inp rotection against viral disease(Rich & Zhang, 2003) .



During the cell cycle, re p lication of DNA occurs .

The DNA double heli x unwinds through thework of an enzyme called a helicase .



The section that o p ens u p is called are p lication fork, because as you can seefrom this model, it resembles a fork .

Another enzyme, DNA polymerase addscom p lementary nucleotides to the parentstrands of DNA

Then, DNA ligase hel p s to wind thestrands back u p into a double heli x.

Lec 3. DNA structure, function and metabolism

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