Problems nonono

8/9/2019 Problems nonono

http://slidepdf.com/reader/full/problems-nonono 1/5

20-1 DNA Supercoiling

1. Why do type II topoisomerase enzymes require ATPwhereastype I topoisomerases do not?

Topoisomerase I reactions are driven by the free energy change ofDNAshifting from a supercoiled conformation to a relaxed conformation, sonoexternal source of free energy is needed. The enzyme merelyaccelerates areaction that is already favorable. Topoisomerase II reactions involve

moreextensive mechanical intervention because both strands of the DNAarecleaved and held apart hile another segment of DNA passes throughthebrea!. This process re"uires the free energy of AT#, since it is notthermo$dynamically favorable on its on.

3. A variety o !ompounds inhi"it topoisomerases. #ovo"io!inis an anti"ioti! and$ %i&e !ipro' o(a!in$ it inhi"its )#A *yrase.)o(oru"i!in and etoposide are anti!an!er dru*s that inhi"iteu&aryoti!topoisomerases. What properties distin*uish the anti"ioti!srom the anti!an!er dru*s?

Novobiocin and cipro%oxacin are useful as antibiotics because theyinhibitpro!aryotic DNA gyrase but not eu!aryotic topoisomerases. They can!ill disease$causing pro!aryotes ithout harming host eu!aryoticcells. Doxorubicinand etoposide inhibit eu!aryotic topoisomerases and can be used asanticancer drugs. Although these drugs inhibit topoisomerases fromboth cancercells and normal cells, cancer cells have a higher rate of DNAreplicationand are more susceptible to the e&ects of the inhibitors than arenormal cells.

'($' The DNA )eplication *achinery

+. ,emi!onservative rep%i!ation is shown in i*ure 20-1. )rawa dia*ram that i%%ustrates the !omposition o )#A dau*htermo%e!u%es

or the se!ond$ third$ and ourth *enerations.

. )#A rep%i!ation o the !ir!u%ar !hromosome in /. !o%i "e*inswhen a protein !a%%ed )naA "inds to the rep%i!ation ori*in onthe)#A. )naA initiates rep%i!ation on%y when the )#A that!onstitutesthe )#A rep%i!ation ori*in is ne*ative%y super!oi%ed. Why?

Negatively supercoiled DNA is more easily unound, and thus theseparationof the DNA strands occurs more easily, hich facilitates thereplicationprocess.

. )#A he%i!ases !an "e !onsidered to "e mo%e!u%ar motorsthat !onvert the !hemi!a% ener*y o #TP hydro%ysis intome!hani!a%ener*y or separatin* )#A strands. The "a!teriopha*e T*enome en!odes a protein that assem"%es into a he(ameri!rin* withhe%i!ase a!tivity.

a In order or the he%i!ase to unwind )#A$ it requires twosin*%e-stranded )#A tai%s at one end o a dou"%e-stranded)#A se*ment. owever$ the he%i!ase appears to "ind to on%yone o the sin*%e strands$ apparent%y "y en!ir!%in* it. Is this!onsistent with its a"i%ity to unwind a dou"%e-stranded )#Ahe%i(?" 4ineti! measurements indi!ate that the T he%i!ase movesa%on* the )#A at a rate o 132 "ases per se!ond. The proteinhydro%yzes 5 dTTP per se!ond. What is the re%ationship"etween dTTP hydro%ysis and )#A unwindin*?! What does the stru!ture o T he%i!ase su**est a"out itspro!essivity?

a +es. y moving along a single DNA strand, the helicase can act asaedge to push apart the double$stranded DNA ahead of it." The free energy of dTT# hydrolysis is similar to the free energy of

AT# hydrolysis. -ach hydrolysis reaction drives the helicase along toto three bases of DNA.! The T helicase is probably a processive enzyme. Its hexamericringstructure is reminiscent of the clamp structure that promotes theprocessivityof DNA polymerase /see 0ig. '(12'3.

11. Temperature-sensitive mutations are de6ned as mutationsthata%%ow a protein to un!tion at a %ow temperature thepermissivetemperature "ut not a hi*h nonpermissive temperature.Temperaturesensitive mutations in some rep%i!ation proteins resu%t in theimmediate!essation o "a!teria% *rowth7 or other mutant proteins$*rowth !omes

to a ha%t more *radua%%y when the "a!teria are e(posed to anonpermissivetemperature. What happens to )#A rep%i!ation and "a!teria%*rowth when the temperature is sudden%y in!reased and thetemperature-sensitive mutation is in a he%i!ase and " )naA seePro"%em ?

a DNA replication /and hence bacterial groth3 halts immediatelyatthe nonpermissive temperature because the DNA cannot be unoundahead of the replication for! in the absence of the helicase.

" acterial groth slos and then stops because the role of DnaA istolocate the replication origin. 4hen the temperature shiftsto the nonpermissive temperature, DNA replication already under ay

isnot a&ected /those cells can complete cell division3, but anotherround of replication cannot begin in the absence of functioning DnaA.

13. In the !hain-termination method o )#A sequen!in*des!ri"ed in ,e!tion 3-5$ the 4%enow ra*ment o )#Apo%ymeraseI see i*. 20-11 is used to synthesize a !omp%ementarystrand usin*sin*%e-stranded )#A as a temp%ate. A%on* with a primer andtheour d#TP su"strates$ a sma%% amount o 2$ 3-dideo(ynu!%eosidetriphosphate dd#TP is added to the rea!tion mi(ture. Whenthe



dd#TP is in!orporated into the *rowin* nu!%eotide !hain$po%ymerizationstops. /(p%ain.

The ddNT# lac!s the 5$ hydroxyl group that serves as the attac!ingnucleophile for the incoming dNT#.

1+. Whi!h eu&aryoti! )#A po%ymerase wou%d you e(pe!t tohave *reater pro!essivity8 po%ymerase 9 or po%ymerase :?/(p%ain.

DNA polymerase 6 ould need to have greater processivity because itsynthesizes the leading strand continuously.#olymerase 7 can be less processive because it synthesizes only ashort DNA segment at the start of each 8!aza!i fragment.

1. )es!ri"e the ways in whi!h a !e%% minimizes thein!orporationo mispaired nu!%eotides durin* )#A rep%i!ation.

0irst, the cell contains roughly e"ual concentrations of the four

deoxynucleotidesubstrates for DNA synthesis9 this minimizes the chance for anoverabundantdNT# to ta!e the place of another or for the rong dNT# to ta!ethe place of a scarce dNT#. Second, DNA polymerase re"uiresaccurate pairingbeteen the template base and the incoming base. Third, the 5:$;<exonucleaseproofreads the nely formed base pair. 0ourth, the removal of the)NA primer and some of the ad=acent DNA helps minimize errorsintroducedby primase and by the DNA polymerase at the ;: end of a ne DNAsegment.0inally, DNA repair mechanisms can excise mispaired or damagednucleotides.

2>. A reaction mixture contains puri?ed DNA polymerase, the

four dNT#s, and one of the DNA molecules hose structures arerepresented here. 4hich reaction mixture produces ##i @

##i is the product of the polymerization reaction catalyzed by DNApolymerase. This reaction also re"uires a template DNA strand and aprimerith a free 5: end.a There is no primer strand, so no ##i is produced." There is no primer strand, so no ##i is produced.! ##i is produced.d No ##i is produced because there is no 5: end that can beextended.e ##i is produced. ##i is produced.

21. Why wou%d it not ma&e sense or the !e%% to wait to!om"ine

;&aza&i ra*ments into one !ontinuous %a**in* strand unti%theentire )#A mo%e!u%e has "een rep%i!ated?

The DNA molecule /chromosome3 is much longer than an 8!aza!ifragment and must be condensed and pac!aged in some ay to ?tinsidethe nucleus /in a eu!aryote3 or cell /in a pro!aryote3. If the cell aiteduntilthe entire DNA molecule had been replicated, the nely synthesizedlaggingstrand, in the form of many 8!aza!i fragments, might already bepac!aged

and inaccessible to the endonuclease, polymerase, and ligasenecessary toproduce a continuous lagging strand.

23. The me!hanism o E. coli )#A %i*ase invo%ves the transero the adeny%y% A<P *roup o #A)1 to the 9-amino *roup o theside !hain o an essentia% %ysine residue on the enzyme. Theadeny%y%*roup is su"sequent%y transerred to the += phosphate *roupo theni!&. The 6rst step is shown in the 6*ure "e%ow. )raw theme!hanism o E. coli )#A %i*ase.

'($5 Telomeres

2+. >ive the name o the enzyme that !ata%yzes ea!h o theo%%owin*rea!tions8a ma&es a )#A strand rom a )#A temp%ate" ma&es a )#A strand rom an #A temp%ate

! ma&es an #A strand rom a )#A temp%ate

a DNA polymerase, " reverse transcriptase or telomerase,! primase or )NA polymerase.

2. In some spe!ies$ >-ri!h te%omeri! )#A o%ds up on itse% to orm a our-stranded stru!ture. In this stru!ture$ our*uanine residues assume a hydro*en-"onded p%anararran*ement with anovera%% *eometry that !an "e represented as



a This is called a "uartet, and it may play a role as a

negative regulator of telomerase activity. Dra the completestructure of this "uartet, including the hydrogen bondsbeteen the purine bases." Sho schematically ho a single strand of four repeating

TTA se"uences can fold to generate a structure ith threestac!ed "uartets lin!ed by TTA loops.

2. An e(periment is !arried out in whi!h the AA@ #Atemp%ate on the te%omerase see i*. 20-1 is mutated. Wi%%there "e a !han*e in the te%omeri! sequen!e as a resu%t othis mutation? /(p%ain.

The resulting telomeres ill have a se"uence complementary to themutated se"uence of the telomerase$associated )NA template. Thisexperimentas important because it established the mechanism of the enzymeand veri?ed the role of the )NA template in extending chromosome

length.

'($B DNA Damage and )epair

31. <utations in the *enes that !ode or repair enzymes !anoten %ead to the transormation o a norma% !e%% into a!an!erous!e%%. /(p%ain why.

4ithout functional DNA repair enzymes, additional mutations mayarise in genes that are involved in regulating cell groth. In theabsence of proper groth controls, cells may begin to proliferate at anaccelerated rate.

33. In eu&aryoti! !e%%s$ a spe!i6 ! triphosphatase !%eavesdeo(y-B-o(o*uanosine triphosphate o(o-d>TP to o(o-d><P 1 PPi .What is the advanta*e o the rea!tion?

The triphosphatase removes nucleotides containing the modi? edbasebefore they can be incorporated into DNA during replication.

3+. What !han*e does the methy%ation o a *uanine residuema&e in the su!!eedin* *enerations o )#A?

The OC$methylguanine produced by the methylation of guanineproducesa residue that can base pair ith either cytosine or thymine. If theOC$methylguanine residue base pairs ith thymine,

3. The !ompound +-"romoura!i% is a thymine ana%o* and !an

"e in!orporated into )#A in the p%a!e o thymine. +-Cromoura!i%readi%y !onverts to an eno% tautomer$ whi!h !an "ase pair with*uanine.The &eto and eno% tautomers ree%y inter!onvert throu*h themovement o a hydro*en "etween an adDa!ent nitro*en ando(y*en. )raw the stru!ture o the "ase pair ormed "y theeno%orm o +-"romoura!i% and *uanine. What &ind o mutation!an+-"romoura!i% indu!e?

romouracil causes an AT to E transition.

3. As dis!ussed in the te(t$ deamination o !ytosineprodu!esura!i%. )eamination o other )#A "ases !an a%so o!!ur. ore(amp%e$deamination o adenine produ!es hypo(anthine.a )raw the stru!ture o hypo(anthine." ypo(anthine !an "ase pair with !ytosine. )raw thestru!ture o this "ase pair.! What is the !onsequen!e to the )#A i this deamination isnot repaired?

! An AT base pair is converted to a E base pair.

51. The a!t that )#A has evo%ved to !ontain the "ases A$ $>$and T ma&es the )#A mo%e!u%e easy to repair. or e(amp%e$deaminationo adenine produ!es hypo(anthine$ deamination o *uanineprodu!es (anthine$ and deamination o !ytosine produ!esura!i%.Why are these deaminations repaired qui!&%y?

All of these deaminations produce bases that are foreign to DNA9therefore,

they can be "uic!ly spotted and repaired before DNA has replicatedandthe damage is passed on to the next generation.

53. The adenine ana%o* 2-aminopurine shown "e%ow is apotent muta*en in "a!teria. The 2-aminopurine su"stitutesoradenine durin* )#A rep%i!ation and *ives rise to mutations"e!ause it pairs with !ytosine instead o thymine. ,tru!tura%studiesshow that there is an equi%i"rium "etween a Eneutra% wo""%eF"ase pair so-!a%%ed "e!ause it is the dominant stru!ture atneutra%p and a Eprotonated WatsonGri!&F stru!ture$ whi!h ormsat%ower p.



a Two hydro*en "onds orm "etween !ytosine and 2-aminopurinein the Eneutra% wo""%eF pair. )raw the stru!ture o this"ase pair." At %ower p$ either the !ytosine or the 2-aminopurine !an"e!ome protonated. The two protonated orms are inequi%i"riumin the Eprotonated WatsonGri!&F "ase pair stru!ture7 theproton essentia%%y Eshutt%esF rom one "ase to the other inthepair$ and the hydro*en "ond is maintained. )raw the twopossi"%e stru!tures or the "ase pair$ one with a protonated2-aminopurine$ and one with a protonated !ytosine.

5+. What is the most !ommon )#A %esion in individua%s withthe disease (eroderma pi*mentosum?

*ost li!ely, the thymine1thymine dimer, since this lesion forms uponexposure of the DNA to ultraviolet light.

5. A strain o mutant "a!teria% !e%%s %a!&s the enzyme ura!i%-)#A *%y!osy%ase. What is the !onsequen!e or the or*anism@

The mutant bacteria are unable to repair deaminated cytosine/uracil3.In these cells, the rate of change of E base pairs to AT base pairs ismuchgreater than normal.

5. )urin* rep%i!ation in E. coli $ !ertain types o )#A dama*e$su!h as thymine dimers$ !an "e "ypassed "y )#A po%ymeraseH.This po%ymerase tends to in!orporate *uanine residuesopposite thedama*ed thymine residues and has a hi*her overa%% error ratethanother po%ymerases. Thymine dimers !an "e "ypassed "y otherpo%ymerases$su!h as )#A po%ymerase III whi!h !arries out most )#A

rep%i!ation in E. coli . )#A po%ymerase III in!orporatesadenineresidues opposite the dama*ed thymine residues$ "ut mu!hmores%ow%y than )#A po%ymerase H. Po%ymerase III is a hi*h%ypro!essiveenzyme$ whereas po%ymerase H adds on%y GB nu!%eotides"eore disso!iatin* rom the )#A. /(p%ain how )#Apo%ymerases IIIand H to*ether !arry out the eJ !ient rep%i!ation o @H-dama*ed)#A with minima% errors.

DNA polymerase III replicates DNA until a thymine dimer isencountered.#olymerase III is accurate but cannot "uic!ly bypass the damage.#olymerase F, hich can more "uic!ly proceed through the damagedsite, does so, but at the cost of misincorporating rather than Aopposite

T. Thus, replication can continue at a high rate. The tendency for DNApolymerase F to continue to introduce errors is minimized by its loprocessivity Soon after passing the thymine dimer, it dissociates, andthemore accurate polymerase III can continue replicating the DNA ithhigh?delity.

'($; DNA #ac!aging

+1. The per!enta*es o ar*inine and %ysine residues in the

histoneso !a% thymus )#A are shown in the ta"%e "e%ow. Why dohistones have a %ar*e num"er o Kys and Ar* residues?

The side chains of lysine and arginine residues have high pK valuesand are positively charged at physiological pG. The positively chargedgroups can form ion pairs ith the negatively charged phosphategroupson the bac!bone of the DNA molecule.

+3. )raw the stru!tures o the side !hains that !orrespond to

theo%%owin* histone modi6 !ations8 a a!ety%ation o Kys$ "phosphory%ationo ,er$ ! phosphory%ation o is$ d methy%ation o Kys$ and e methy%ation o Ar*. ow do these modi6 !ations!han*ethe !hara!ter o their respe!tive amino a!id side !hains?

++. )#A methy%ation requires the methy% *roup donorS-adenosy%methionine$ whi!h is produ!ed "y the!ondensation o methionine with ATP. The su%onium ion=s methy% *roup isused inmethy%-*roup transer rea!tions.



a The demethy%ated S -adenosy%methionine is thenhydro%yzed toprodu!e adenosine and a nonstandard amino a!id. )raw thestru!ture o this amino a!id. ow does the !e%% !onvert this!ompound "a!& to methionine to re*enerate S

-adenosy%methionine?" The proper re*u%ation o *ene e(pression requiresmethy%ationas we%% as demethy%ation o !ytosine residues in )#A. I ademethy%ase !arries out a hydro%yti! rea!tion to restore!ytosineresidues$ what is the other rea!tion produ!t?

a The nonstandard amino acid is homocysteine, hich can accept amethyl group donated by methyl$tetrahydrofolate to regeneratemethionine

" The other product is methanol, EG58G.

+. Proteins destined or the proteasome are ta**ed withu"iquitinas des!ri"ed in ,e!tion 12-1. ow does this modi6 !ation!omparewith the modi6 !ation o a histone with u"iquitin? Areu"iquitinatedhistones mar&ed or proteo%yti! destru!tion "y theproteasome?

No, histones modi? ed ith ubi"uitin are not mar!ed for proteolyticdestruction by the proteasome because the amino acid side chains of

thehistone proteins have only one ubi"uitin attached. In order to entertheproteasome for degradation, a chain of at least four ubi"uitinmolecules isre"uired /see Section 2'$23.

+. )urin* sperm deve%opment$ a"out +L o the !e%%=s )#A isasso!iated with sma%% proteins &nown as protamines ratherthanhistones. ProtamineG)#A !omp%e(es pa!& more !ompa!t%ythanhistoneG)#A !omp%e(es.a /(p%ain the advanta*e o rep%a!in* histones withprotaminesdurin* sperm deve%opment." What type o *enes wou%d you e(pe!t to 6 nd in the

remainin* nu!%eosomes? Hint

8 these *enes are nottrans!ri"edunti% ater erti%ization.

a ecause the protamine1DNA complexes occupy less volume thannucleosomes, DNA can be more easily pac!ed into the small volumeof the sperm nucleus." DNA in nucleosomes is less compact and therefore moreaccessiblefor transcription, so one ould expect nucleosomal DNA to containgenes that ould need to be expressed immediately folloingfertilization,that is, genes essential for early embryogenesis.

Documents

Problems nonono