Quiz 2

Question 1

leading strand vs. Lagging strand

Answer 1

leading strand is continuous and DNA Polymerase chases the replication fork in 5->3’ direction from an exposed 3’ hydroxyl (-OH). The first RNA primer is synthesized by (DnaG) primase
Lagging strand has discontinuous synthesis and needs to wait for upstream DNA 3’-OH to be exposed. needs many primers

Question 2

what are Okazaki fragments and how do they compare in prokaryotes and eukaryotes

Answer 2

=newly synthesized stretches of DNA on the lagging strand

• in prokaryotes, usually 1000-2000 nucleotides
• in eukaryotes, usually 100-400 nucleotides
• Length due to processivity

Question 3

what does RNA Polymerase usually need to bind to the DNA so it can provide a short primer in which DNA can expand upon

Answer 3

Short DNA sequence trimer motif (GTA in E. coli.)

Question 4

what enzymes remove RNA primers?

Answer 4

RNase H and 5’ exonuclease

Question 5

describe the removal of the RNA template/primer to copied DNA strand

Answer 5

RNase H only bind to RNA bound to DNA and removes almost all the primer except for one which is removed by 5’exonuclease. DNA Pol then fills in the gap and DNA ligase seals the nick

Question 6

compare the type of ligase used for E coli DNA vs T4 virus and eukaryote DNA ligase

Answer 6

E. coli ligase uses NAD+ as a cofactor

Eukaryotes use Mg+ATP

Question 7

describe the structure/role of DNA helicase

Answer 7

unwinds DNA at the replication fork

• they are hexameric proteins that encircle one of the DNA strands
• highly processive: unwind multiple bases, don’t fall off.
• needs ATP to break the H-bonds between bases and to move along the DNA
• they are loaded onto DNA
• they activate primases/primers

Question 8

how is unwound DNA stabilized ?

Answer 8

by single stranded DNA binding proteins (SSB)

• the first one that binds facilitates others to bind and protect DNA, getting it ready for replication
• SSBs bind to the phosphodiester backbone and stack with nucleotide bases
• SSBs dont form Hydrogen bonds with the ssDNA like other DNA binding proteins do

Question 9

what is the role of topoisomerase in DNA replication

Answer 9

DNA topoisomerase 11 relieves positive supercoiling caused by replication by cutting both DNA strands and holding onto them both.
-acts on unreplicated DNA ahead of the replication fork to reduce linking number (cut about every 10 base pairs)

Question 10

list the steps in how DNA replisome proteins function at the replication fork to duplicate DNA

Answer 10

1. Helicase unzips DNA with ATP
2. DNA topoisomerase II unwinds
3. SSBs bind & protect DNA for replication
4. Primase adds in temporary template
5. DNA polymerase (III) extends DNA
6. DNA polymerase I replaces gaps
   where RNase H removed RNA primer
   and seals nicks in DNA (nick ligation with ligase)

Question 11

How are DNA polymerase isoforms distinguished ?

Answer 11

by enzyme kinetics (how fast they work), subunit composition, specific function and abundance

Question 12

compare DNA Pol 1 and DNA Pol III isoforms (Prokaryotes)

Answer 12

DNA Pol III: highly processive since it replicates
4.6 Mb E. coli genome from only 2 replication forks

DNA Pol I: not processive (20-100nt/binding), removes upstream RNA from Okazaki fragment, fills in gap left by RNase H, has own built in 5′ exonuclease activity

• Both these have proof-reading ability, other 3 isoforms do not (repair only)
• Prokaryotes have 5 DNA Pol isoforms, 2 “hi-fi” for replication, 3 for repair only

Question 13

Compare the three replication DNA Polymerase isoforms in eukaryotes

READING p269-278

Answer 13

3 genome replicating polymerases: DNA Pol α, ε and δ
- DNA Pol α/primase H starts off
with primase activity then DNA pol
activity (but has low processivity)
* DNA Pol α does both primer and DNA lay down but its slow so it switches)
-Replication shifts into ‘top gear’ with
highly processive DNA pol ε and δ
(this is called “polymerase switching”)

Question 14

what is the function of the sliding clamp for DNA replication?

Answer 14

the sliding clamp encircles DNA, binds to DNA Pol and keeps it from diffusing away and on track

• DNA Pol dissociates from 3′-OH every 20-100 bases but sliding clamp prevents loss
• vastly improves processivity of DNA Pol

Question 15

how/when does the sliding clamp-DNA Pol complex separate?

Answer 15

=dissociates when it hits double-stranded DNA
-DNA Pol recognizes dsDNA, active site has low affinity for dsDNA (as opposed to high affinity for RNA primer:DNA template junction)
-DNA Pol undergoes conformation change,
sliding clamp has low affinity for this DNA Pol’
-DNA Pol′ diffuses away but sliding clamp (PCNA)
remains for nucleosome assembly role

Question 16

how does the sliding clamp get put on/taken off the DNA strand

Answer 16

• Sliding clamp loaders bind and use ATP to crack open sliding clamp ring
• The ‘open’ hexamer can then encircle a DNA strand, recruit, bind DNA Pol
• When DNA Pol , nucleosome packing done, sliding clamp loaders also remove sliding clamps from the DNA strand (so DNA bases can pair)
• Presence of primer:template junction serves as recruitment signal for sliding clamp (during replication, also during DNA repair)
• DNA Pol and sliding clamp loaders compete for same binding domain on the sliding clamp itself -> so loader can’t interfere with active DNA Pol

Question 17

describe the steps (a-e) of how the slide clamp is loaded onto the DNA strand

Answer 17

(a) Slide clamp loader protein (‘З-like’ protein) senses
primer:template junction
(b) binding of ATP, conformational change
(c) binds to protein:protein interaction domain of
sliding clamp protein, breaks hexameric ring
(d) recognizes p:t junction, slips ring onto DNA strand
(e) DNA binding causes ATP hydrolysis by altering
conformational shape, causing dissociation,
DNA is left encircled by clamp protein

Question 18

what is the name of the Slide clamp loader protein in pro and eukaryotes

Answer 18

Slide clamp loader protein is γ-complex in E. coli,

Replication Factor C (RF-C) in eukaryotes

Question 19

describe the makeup of the DNA Pol III holoenzyme

Answer 19

a complex of DNA Pol, clamp loader, & sliding clamp

• E. coli DNA replication can be coordinated by physically linking enzymes (DNA Pol III holoenzyme)
• Tau (τ)-protein links clamp loader with two DNA Pol III subunits (1 for leading + 2 for lagging strand synthesis)

Question 20

what is faster, DNA Pol III or primase?

https://www.youtube.com/watch?v=I9ArIJWYZHI

Answer 20

DNA Pol III is faster than primase, so DNA Pol III is always
waiting for primase to dissociate (get out of the way)

Question 21

DNA Pol III replicates as fast as helicase unwinds it, but only when….?

Answer 21

only when helicase interacts with τ -protein
(When helicase is not binding the τ –protein, activity falls 10-fold, allowing DNA Pol to catch up and preventing “helicase runaway”)
*Interactions of helicase and τ –protein help coordinate DNA Pol and helicase speed

Question 22

how do Interactions of helicase and primase regulate Okazaki fragment length ?

Answer 22

• Helicase also has protein-protein interaction with DNA primase, Interaction stimulates primase activity 1000 X’s
• Stronger association gives more primers, shorter Okazaki fragments
• Weaker gives less primers, longer Okazaki fragments

Question 23

replicon = ?

Answer 23

all DNA replicated from 1 origin of replication (circular chromosome=1 replicon)

Question 24

how does replication start? Origins of replication …

Answer 24

=Initiator protein binds replicator DNA motif at origin of replication to start DNA replication

-Need replicator (DNA sequence motif sufficient to
direct initiation of DNA replication), part of origin of
replication but not always be the entire origin in
eukaryotes (may need other DNA sequences)
-Need initiator (protein) to recognize DNA motif in replicator to activate replication
(uses ATP, recruits other proteins in to bind it or other DNA
structures, e.g., primer:template junction)

Question 25

Known replicator sites share 2 common features READING= p278-292

Answer 25

1. Binding site for initiator 2. AT-rich DNA motif that unwinds fairly easily…. * Replicator DNA sequences have binding & unwinding, initial DNA synthesis sites

Question 26

what are the two main jobs of Initiator proteins?

Answer 26

1. Bind to replicator regions’ DNA sequence | 2. Recruit proteins/replisome units for DNA replication

Question 27

what is the additional role of initiator proteins in prokaryote DNA? (E. coli)

Answer 27

Facilitate unwinding of DNA

Question 28

compare initiator proteins in E coli and eukaryotes

Answer 28

* E. coli DnaA protein binds 9 & 13 mer regions, helps unwind DNA, recruits proteins * origin replication protein complex (ORC) is eukaryotic equivalent to DnaA, binds to origin element, helps recruit helicase (+ATP…) & all other proteins, BUT does not unwind DNA

Question 29

explain how the DnaA initiator protein binds and starts the rep. process

Answer 29

DnaA bind oriC area and DnaA complex ‘melts’ the DNA repeats to form an “open complex“ (ATP also needed) -Helicase + loading protein binds to melted area to form a "prepriming complex“, loading protein drops off and activates helicase -In the presence of SSB plus DNA gyrase, helicase unwinds the DNA further. -Helicase recruits DnaG primase which makes a primer -Helicase action displaces the DnaA initiator protein as oriC is fully unwound

Question 30

To avoid catastrophic gene changes, only replicate chromosomes once during S phase Rereplication of any DNA is difficult to fix, what may happen when attempting to fix this

Answer 30

Attempts to fix this often leads to gene duplication, increased expression that causes unwanted biological response -Incomplete duplication followed by premature segregation (pulling apart of chromosomes) breaks DNA

Question 31

how many replication origins are there on our chromosomes and how spaced out are they? when does it get inactivated?

Answer 31

- one replication origin every ~30kb - 1500-8200 replication origins on our chromosomes - the origin gets inactivated once it starts replicating, or is (passively) replicated from another origin so **No origin can initiate replication once it has been replicated itself (Ensures requirement of replicating chromosome once per cycle! )

Question 32

what is the first step in the | initiation of replication in eukaryotes

Answer 32

helicase loading

Question 33

Two phases during replication?

Answer 33

1. Identification, selection, ORC initiator binding to replicator during G1 phase (before S phase) 2. Origin activation during S phase to trigger helicase (McM2-7) activity , DNA Pol recruitment

Question 34

ORC has 2 helicase loaders which are?

Answer 34

Cdc6 and Cdt1 | *Cdc6/ORC/Cdt1 (Prereplicative complex) assembles in G1 phase and waits for S phase signal before actoin

Question 35

S phase signal is ??

Answer 35

S phase signal is phosphorylation activation of helicase binding cascade

Question 36

Two protein kinases activated in S phase?

Answer 36

CDK =(Cyclin Dependent Kinase) | DDK= (Dbf4-Dependent kinase)

Question 37

CDK and DDK phosphorylate what? what is the result?

Answer 37

DDK phosphorylates helicase, CDK phosphorylates CMG helicase complex(Sld2/3, Dpb11 -> facilitating GINS, Cdc45 binding to helicase) * Cdc6, Cdt1 helicase loader is even proteolyzed due to phosphorylation * DNA Pol α/primase, then other DNA Pol are also recruited, preceding 1st RNA primer

Question 38

CDKs have 2 key functions;

Answer 38

1) activate helicases to start DNA replication | 2) also inhibit new helicases formation

Question 39

how is CDK activity regulated to allow only a single round of replication during each cell cycle?

Answer 39

CDK activity is regulated – low during G1 phase (allows helicase loading, but not activation) CDK activity high during S phase (G2 &M also) (causes helicase activation, stops helicase loading) *ensures only 1 chance for helicase loading in G1 phase with only 1 chance to activate helicase in S phase

Question 40

what allows the proper segregation of daughter | chromosomes after replication?

Answer 40

- Both circular and large linear chromosomes need to be decatenated or untangled/unknotted, respectively - Type II topoisomerases finish circular DNA replication by decatenation

Question 41

large linear chromosomes have another problem after replication, what is it?

Answer 41

Lagging-strand synthesis is unable to copy the extreme ends of linear chromosomes because there is a requirement for RNA primer -therefore because primase runs out of space to bind last part of DNA, 1 chromosome is incompletely replicated

Question 42

how does telomerase fix the problem of the lagging strand being unable to copy the ends of the linear chromosome?

Answer 42

because it is a novel DNA polymerase that does not require an exogenous template and uses its ribonucleoprotein component to extend the 3′ end with reverse transcriptase activity -Telomerase uses its own RNA to bind to DNA, then use 3′OH of ssDNA to extend DNA with a telomerase subunit that is a reverse transcriptase (uses RNA as template for making DNA) -Newly added 3′ end DNA gives primase room to add RNA template, allow DNA Pol III to extend the DNA fragment again -Still leaves extra 3′ end overhang but this can extend telomeres

Question 43

Unregulated telomere extension is unwanted so how is it regulated? describe the proteins in yeast and humans

Answer 43

=Telomere-binding proteins regulate telomerase activity and tail -Telomeres bound by ‘shelterins’ which regulate length by governing telomerase-extension activity -In yeast, Cdc13 binds ssDNA, recruits telomerase. it also has Rap1 (dsDNA-binder), recruits Rif1, Rif2 -Humans also have specialized Telomere Repeat binding Factors (TRFs) like yeast but unlike yeast, ssDNA binder POT1 inhibits activity of telomerase (more shelterin protein/POT1 = less telomere extension)

Question 44

how do Telomere-binding proteins protect chromosome tails? READING: pg 293-310

Answer 44

Human telomeres apparently form T-loops with help of TRF2 (this works in vitro) -3′ ssDNA folds back on itself, invades dsDNA region of telomere, binds DNA and then is not recognized as dsDNA break (is protected as it is hidden)

Question 45

Mutations in DNA are almost always negative and need to be reversed so cells must do what two things?

Answer 45

Cells must 1) detect damaged DNA 2) repair and restore DNA code

Question 46

Mutations can arise due to DNA replication inaccuracy or chemical damage and can either ?

Answer 46

1. change DNA code forever-> Allow use of DNA… but changes genetic code (cytosine deamination to uracil ->C:G to T:A…) 2. make DNA unusable-> Block use of DNA in transcription, replication (DNA break, thymine dimer) -Needs to be fixed before 2nd round of DNA replication or it is permanent

Question 47

why is there no cure for cancer?

Answer 47

because they are so many types of cancer and it changes from person to person and even within the same person - cancer is not a single mutation but a whole bunch of possible mutations - instead you need to find a million cures for a million different kinds of cancer - cancer has found ways to cheat death in all its forms

Question 48

Simplest mutations are changes to single bases: two types of substitutions?

Answer 48

Transitions (pyrimidine-pyrimidine, purine-purine) Transversions (purine to pyrimidine & vice versa)

Question 49

Insertions & deletions are more complex mutations that are caused by?

Answer 49

Insertions caused by Transposons (‘jumping genes’) Deletions caused by recombination reactions -These can cause frameshift mutations, ± 1 or 2 bp (can be due to DNA intercalating mutagens, cause ‘slippage’ during DNA replication)

Question 50

describe how the “mismatch repair system” is the last line of defence for DNA replication (in E coli)

Answer 50

-Improves fidelity 2-3 orders of magnitude MutS (=MutationScanner) in E. coli repairs mismatched DNA bases and has two functions: 1) Rapidly scan genome for mismatches (backbone distortion induced by mispairing) 2) Correct repair of mismatch on daughter, not parental template strand

Question 51

how does MutS act to recruit proteins that clip, | degrade, refill & seal mismatched area?

Answer 51

-first, MutS binds to mismatch distortion and undergoes conformational change (MutS′) -then, it recruits MutL(oader), MutH(ealer) -MutL activates MutH, an endonuclease that nicks DNA, UrvD helicase (UV response D) unwinds DNA -An exonuclease removes DNA to clear area -DNA Pol III fills in new gap, nick sealed by a DNA ligase to finish repair

Question 52

how does MutS′ know which strand is the parent and daughter? (in bacteria)

Answer 52

Transient methylation of Adenine in parental DNA by DexoyAdenosine Methylase (DAM) which acts on GATC about every 256bp -Daughter DNA duplex only methylated solely on parental strand for short time after replication so MutS′ must act before DAM methylase catches up and methylates new daughter DNA… -MutH recognizes hemimethylated DNA so MutL & MutS can activate the endonuclease activity (MutH binds nonmethylated DNA strand) ( our DNA is not methylated like bacteria’s)

Question 53

what are the different exonucleases recruited depending on what side of MutS makes nick

Answer 53

If nick is on 5′ side of mismatch: Exonuclease VII (5′-> 3′) removes DNA between the nick caused by MutH and the site of the mismatch If nick is on 3′ side of mismatch: Exonuclease I (3′-> 5′) removes DNA between the nick caused by MutH and the site of the mismatch

Question 54

DNA is Damaged Spontaneously by ? and ?

Answer 54

Hydrolysis & Deamination

Question 55

how is DNA damaged by water?

Answer 55

->DNA is damaged by the action of water that causes deamination - C->U transversion happens in mammalian cells at rate of 100 events/day (most common) -Adenine, Guanine susceptible too -Uracil is unnatural in DNA, makes bond with adenine (instead of proper C-G!) -DNA is also depurinated by water: Guanine depurinated to nothing (abasic site causes loss of a base in the DNA helix)

Question 56

why isnt uracil used in DNA?

Answer 56

because there is no way to know if it is there due to deliberate inclusion or because a cytosine was deaminated!

Question 57

Deamination of DNA bases can be chemical too (nitrosamine ->forms N oxides) Nitrosamine comes from ??

Answer 57

comes from nitrates and nitrites that are common food preservatives (also in beer, tobacco, fried bacon, cured meats, nonfat dry milk)

Question 58

deamination of what gives normal base (T) ?

Answer 58

-5-methylcytosine (epigenetics) deamination gives normal base (T) so these are mutation “hotspots”

Question 59

how is DNA Damaged by alkylation

Answer 59

Alkylation transfers methyl/ethyl groups to bases, phosphates in backbone O6-methylguanosine leads to a G:CA:T mutation

Question 60

how is the very mutagenic oxoG (7,8-dihydro-8-oxoguanine) made?

Answer 60

made by reactive oxygen species (oxidation) and can pair with both adenine and cytosine

Question 61

what is the most common mutation in cancer

Answer 61

oxoG (7,8-dihydro-8-oxoguanine)

Question 62

how is DNA damaged by radiation?

Answer 62

UV radiation causes thymine dimer formation, stops replication of DNA Radiation causes dsDNA strand breaks, stops replication

Question 63

what are Base analogs and how can they cause DNA mutation

Answer 63

Base analogs chemically resemble the proper DNA bases, but can be improperly incorporated, leading to improper base pairing -> mutation

Question 64

what are Intercalating agents and how can they cause DNA mutation READING: pg 313-324

Answer 64

Intercalating agents are flat, can slip between bases of DNA | -Can cause deletion/insertion of a base or few bases (frameshift)

Question 65

what are the 4 categories of DNA repair systems

Answer 65

1. reversal of damage by enzyme (most simple repair) 2. Excision repair = damage removed, not reversed (more complex) - >Undamaged DNA becomes template for DNA polymerase (Either a single, or a stretch of nucleotides is replaced) 3. Recombinational =double-strand break: repair uses info from 2nd copy of chromosome *most cytotoxic DNA damage 4. If replication blocked by damaged base, translesion DNA polymerase copies across it without proper base-pairing (last-chance system, mutagenic)

Question 66

describe photoreactivation

Answer 66

=Best example of simple repair by reversal - Thymine bases damaged by UV radiation -> cyclobutane ring formed - DNA photolyase can use light energy to break ring’s covalent bonds

Question 67

role of methyltransferase in simple DNA repair by reversal? where is the enzyme expressed?

Answer 67

methyltransferase removes the methyl group from O6-methylguanine (from alkylation reaction)->but it destroys the methyltransferase! -Enzyme is expressed mainly in liver ->where metabolism of toxic nitrosamines happens (beer, bacon, smokes)

Question 68

how can Alkylation of adenine and cytosine be reversed

Answer 68

by AlkB in newly discovered pathway - AlkB carries out oxidative demethylation reaction and will attack methyladenine and methylcytosine * Oxidized methyl groups are released as formaldehyde (toxic by itself…)

Question 69

how do Base excision repair enzymes remove damaged bases?

Answer 69

remove damaged bases by a base-flipping mechanism: -DNA glycosylases scan along minor groove until damaged base encountered (base flipped out), then glycosylase active site binds base (has two chances to act)

Question 70

describe how DNA glycosylase has one more chance if it fails to remove damaged base before replication

Answer 70

- oxoG can mispair with A, but “fail-safe” repair removes A and replaces with proper base (C) - OR, 5-methylcytosine is deaminated to thymine (natural base), gives T:G pairing instead but repair system replaces the T with a C (assumes it is an error)

Question 71

nucleotide excision repair enzymes differ from DNA gycosylase in that they do ? rather than recognizing damaged bases

Answer 71

recognize distorted DNA (thymine dimer, bulky adduct)

Question 72

describe the steps in how Nucleotide excision repair enzymes (Uvr_) remove and replace a stretch of bad DNA (5 steps)

Answer 72

- UvrA2B complex scans DNA - UvrA(ssessor) dissociates from UvrB when distortion found - UvrB(inder) opens DNA, recruits UvrC - UvrC(hopper) cuts on either side of UvrB - helicase (UvrD) unwinds DNA, allows DNA polymerase, ligase to replace it

Question 73

what is xeroderma pigmentosum (XP)

Answer 73

mutations in XP protein leading to extreme UV sensitivity | *2000-4000x’s normal rate of skin, eye cancer in X.P.

Question 74

“transcription-coupled repair”

Answer 74

Targets repair enzymes to genes in use ->Critical nucleotide excision repair enzymes prioritize actively transcribed genes (help rescue stalled RNA polymerase) (Transcription-coupled repair systems also exist in prokaryotes)

Question 75

how does DNA recombination help to repair DNA lesions that block replication fork

Answer 75

Undamaged template of the other daughter strand can be used for template -and then damage to parent strand can be fixed like other type 2 excision repairs

Question 76

when/how is ‘Non-Homologous End Joining” (NHEJ) used to repair Double-stranded breaks (DSB) in DNA

Answer 76

when there is only 1 chromosome (early cell cycle) to get info from -(Ku70/80) proteins bind to ends of DNA -DNA protein kinase (DNA-PKcs) recruited, recruit Artemis endo/exonuclease and phosphorylate it (activates) -Artemis-processed ends then joined by ligase -While mutagenic, still the lesser of 2 evils…

Question 77

how does Translesion DNA synthesis enable | replication to go across DNA damage

Answer 77

bypass damage sites, replication continues: -special template-independent DNA Pol IV, V replicate across lesion, then DNA Pol III resumes -Highly error-prone but cell has little choice (replicate or die) -DNA Pol IV, V only expressed when DNA damage is sensed (SOS response – DNA damage destroys LexA repressor protein ->allows expression of these DNA Pol IV, V isoforms and causes activation of Pol V)

Question 78

Eukaryotes bypass and fix lesions by ubiquitinating regular DNA polymerases. what two ways can this happen? READING: p325-338

Answer 78

a) May happen by polymerase switching (a) where stalled replicative polymerase gets ‘Ub tagged’ on its sliding clamp then and swapped out for translesion polymerase b) May happen by “gap filling” (b) where replicative DNA Pol allowed to skip lesion, translesion polymerase fixes it later