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Biochem 3382 > Midterm > Flashcards

Midterm Flashcards

1
Q

Why is DNA a double helix? Why is it right-handed?

A

the hydrophobic bases want to be hidden from water
in order to do this and minimize steric clashes, the bases are kind of shifted and twisted
repeats this over and over gives a double helix
is right-handed because it packs together better that way

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2
Q

What is the spacing between bases? How thick are the bases? What is the actual distance between bases if the backbone is stretched out?

A

0.6nm / 6 A is the the spacing
each base is 0.33nm thick (3.3A)
so there is actually 0.27/2.7A of space between them

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3
Q

How many bp/turn are there in an ideal B-DNA helix?

A

10 bp/turn

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4
Q

How wide is an ideal B-DNA helix?

A

2.4 nm

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5
Q

How much space is there between bases in an ideal B-DNA helix?

A

0.34 nm

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6
Q

Is B-DNA right or left handed?

A

right

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7
Q

How can you tell which is the major or minor groove?

A

it is based on the angle between the deoxyriboses

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8
Q

What is propeller twist?

A

bases in a pair twist in opposite directions to minimizes exposure of the hydrophobic areas

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9
Q

When is propeller twist a problem?

A

when a pyrimidine is on top of a purine or vice versa

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10
Q

What are the 2 ways DNA can deal with propeller twist with a purine and pyrimidine on top of each other?

A

bases can slide relative to each other so that the H bonds etc line up and propeller twist can happen without a steric clash
this is called slide
or
they can slide so they are actually on top of each other, giving more interactions between the purines, the pyrimidines still have propeller twist
this is called roll

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11
Q

What is twist?

A

the way the bases twist about 36 degrees relative to each other

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12
Q

What is shift?

A

shift is movement in the x axis (think back and forth) vs slide which is in the y axis (think side to side)

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13
Q

Explain how the bonds in DNA are labelled

A

alpha is the first P-O
beta is the O-C
gamma is C to the C on the sugar
delta is the next C-C
epsilon is the sugar to the O of the next phosphate
zeta is the O to the P in that phosphate
chi is the N-glycosidic bond (sugar to base)

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14
Q

What are the 2 conformations of chi?

A

can be anti or syn
pyrimidines are always anti
everything in B-DNA is anti

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15
Q

How many degrees of freedom are there in the bonds in DNA?

A

6

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16
Q

What are the 2 major conformations that the phosphate between 2 nucleotides can adopt called? Which bonds do they involve? Which conformation has the top nucleotide in more?

A

BI and BII
involve primarily epsilon and zeta
BII has the top one in more

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17
Q

How many possible dinucleotides are there? How are they written?

A

10

5’ to 3’ of one of top of the other, then 5’ to 3’ of the ones that are stacked opposite them

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18
Q

What is a TRX score?

A

involves the twist, roll and shift
used for ranking the flexibility of dinucleotides
it is the average of the %BII of each half

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19
Q

Which conformation of phosphate appears to be more stable?

A

BI

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20
Q

Explain the relative flexibility of dinculeotides i.e. which ones are more flexible

A

it is generally accepted that GC rich base pairs tend to be more flexible than AT rich base pairs

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21
Q

Explain what Sxy score is and what its used for

A

it is a measure of entropy, is used to measure dinucleotide flexibility
the higher the score, the more flexibility there is

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22
Q

Explain how the BI and BII conformations affect the grooves in protein and thus protein binding

A

BI-rich stretches are less flexible than BII-rich stretches
the minor groove is much more open the the flexible, BII-rich stretches
(the shape of the major groove is also different)
if DNA is more flexible, protein can adapt the structure of the DNA to its surface when it binds, which is going to be more forgiving for proteins trying to bind
ie proteins may not be able to bind to an inflexible region
Overall: BI-rich regions won’t adapt when a protein tries to bind, but BII-rich regions will be able to a bit

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23
Q

What are the 3 forces that influence nucleic acid structure?

A

base stacking
base pairing
ionic interactions
Note: these are for DNA and RNA

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24
Q

Explain how base stacking influences nucleic acid structure

A

van der Waals are partial charge interactions between the rings of bases hold them together
it is sequence-dependent
GC stacking interactions are stronger than AT stacking interactions
i.e. they have a more negative stacking energy (the energy it would take to get them apart)

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25
Q

Explain how dinucleotides are asymmetrical

A

because of right-handed twist when bases are stacked the charges are distributed differently
ie GC/GC is different from CG/CG
or AT/AT is different from TA/TA

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26
Q

Explain how base pairing influences nucleic acid structure and how H bonds are involved

A

the advantage to making H bonds with the other bases instead of with water is cooperativity
when bases are bound together you would need to rip off all 2 or 3 H bonds at once (which is harder) vs with water where you could get rid of them one at a time (easier)

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27
Q

Explain how ionic interactions influence nucleic acid structure

A

the repulsion between negatively charged phosphates is shielded by cations
divalent are better than monovalent
Mg2+ with 6 H20 are the best, the geometry is perfect
(all cation-DNA interactions are mediated by water)

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28
Q

What temperature should you re-anneal DNA strands at?

A

15-25 degrees below the Tm

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29
Q

Are denaturation and renaturation cooperative?

A

yes

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30
Q

Explain how you can measure DNA denaturation/renaturation using UV absorbance

A

all of the bases will absorb UV light
max absorbance is around 260nm
ssDNA has a higher UV absorbance than dsDNA
so you can watch the absorbance increase to see when your DNA is fully denatured

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31
Q

What is Tm?

A

the melting temperature, is the temp at which half of the DNA is denatured i.e. half the bases are paired, half aren;t

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32
Q

How does GC content affect Tm?

A

higher GC content = higher Tm

Note: that the order of bases matters too because of base stacking

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33
Q

How does strand length affect Tm? What about complementarity?

A

longer strand = higher Tm

more complementarity = higher Tm (mismatch = lower Tm)

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34
Q

How do organic solvents affect Tm?

A

they decrease Tm
organic solvents are usually non polar, which makes the solution more hydrophobic and decreases the drive to have the bases in the middle

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35
Q

How does salt concentration affect Tm?

A

increasing salt increases the Tm because it can shield the negative charges in DNA

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36
Q

How does pH affect Tm?

A

generally at a higher pH, you have a lower stability and thus a lower Tm

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37
Q

Describe an A-DNA double helix. What prefers to make this structure?

A 
right-handed 
11 bp/turn (i.e. lower twist) 
has a larger tilt
minor groove is very shallow
major groove is very deep 
when RNA makes double helices with itself, it tends to adopt this type of structure (it can't make B-DNA-like structure because of its extra OH)
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38
Q

Describe a Z-DNA double helix

A

left-handed
12 bp/turn
chi angles are anti for pyrimidines, but syn for purines (i.e. purine ring is over the ribose sugar)
formed in vitro under high salt conditions
usually have sequences that alternate purines and pyrimidines i.e. CGCGCG or CACACAC
binding proteins have been found
thought to be involved in gene regulation
major groove is flat, minor groove is narrow and deep

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39
Q

Explain Hoogsteen base pairing and some motifs it can give rise to

A

purines can make other type of interactions with their corresponding pyrimidines through their major groove side
called H-DNA
can make DNA hinge \
if one region is rich in purines it can form Hoogsteen base pairs with the corresponding strand that is rich in pyrimidines instead of the pyrimidine strand H bonding to the one it normally would
if you have many Gs together, they can H bong together in 4s, with a metal ion in the centre
check textbook for example with Us

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40
Q

Name 6 ways RNA is different from DNA

A 
has a 2'OH
is degraded at high pH 
is single-stranded
uses U instead of T
is shorter
is more likely to have base modifications
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41
Q

Why does RNA often make non-Watson-Crick base pairs?

A

because it folds back on itself so it can cross at different angles and come together in different ways

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42
Q

What is a bulge?

A

one strand has some bases that are not involved in the base pair

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43
Q

What is a loop? What is a hairpin loop?

A

both strands have bases that aren’t paired

hairpin is when its at the end

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44
Q

What is a pseudoknot?

A

kind of have a hairpin loop and then there is a free end that comes back around and base pairs to the loop

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45
Q

What is coaxial stacking?

A

RNA wants to maximize base pair stacking
have 3 hairpin loops, it will arrange so two are sticking out and one has the rest of the strand stacked on top of it
Note: base stacking can occur in single-stranded regions too

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46
Q

Explain some features of 5S rRNA

A

has a hairpin loop and other parts that can interact with proteins, other RNA etc
there is base stacking in the single stranded regions

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47
Q

What are ribose zippers?

A

OH group of the ribose can form H bonds with the OH the ribose on the other backbone
OH groups can also form H bonds with the bases
phosphate groups can also form H bonds
there are many different combinations
H bonding the phosphates helps stabilize their negative charge
i.e. the ribosome has a lot of RNA in it, this can help stabilize the charge so that it can fold together
Note: THESE ARE ONLY IN RNA

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48
Q

What is the A minor motif?

A

only in RNA because it can fold back one itself!

the minor groove side of adenine interacts with a ribose OH

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49
Q

What is the G-ribo motif?

A

only in RNA

G can H bond with ribose of another part of the strand

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50
Q

Can bases involved in A minor and G-ribo motifs also be in Watson-Crick pairs?

A

yes if there is enough room

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51
Q

What is twist (in relation to DNA coiling)?

A

the number of complete turns one strand makes around the axis of the double helix (duplex axis)
RH is positive, LH is negative
for B-DNA T= #bp/10

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52
Q

What kind of supercoiling do you get from underwound RH DNA?

A

negative

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53
Q

What kind of supercoiling do you get from overwound RH DNA?

A

positive

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54
Q

What is another name for interwound writhe?

A

plectonemic writhe

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55
Q

What is writhe?

A

the number of times the duplex axes cross each other

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56
Q

What kind of interwound writhe do you get when you underwind RH DNA?

A

gives RH interwound writhe

which is negative (because its from underwinding)

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57
Q

What is another name for spiral writhe?

A

toroidal writhe

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58
Q

What is spiral writhe?

A

the number of turns the duplex axis makes around the superhelical axis

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59
Q

What kind of spiral writhe do you get when you underwent RH DNA?

A

gives LH spiral writhe

which is negative (because its from underwinding)

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60
Q

What is linking number?

A

the number of times one DNA strand winds around the other
L= T + W
L cannot change unless a strand is broken or the ends of linear DNA are not fixed

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61
Q

What does “relaxed” mean for DNA?

A

no writhe and no tension

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62
Q

What is superhelical density? Does it change?

A

The amount of supercoiling relative to the length of DNA
sigma= (L-L0)/L0
L0 is the L of relaxed DNA
when T= bp/10, sigma is equal to the number of supercoils per B-DNA repeat
doesn’t change unless the linking number changes

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63
Q

Describe Type 1A topoisomerases

A

cut one strand then pass it through the resulting gap
each time it changes the linking number by 1
can only INCREASE the linking number
think of it as introducing positive supercoiling or removing negative supercoiling
they also only relax DNA, will only bring W closer to 0 i.e. if its positively supercoiled they will not act on it
does not require ATP
relies on the favourable process of relieving supercoiling/relaxing DNA

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64
Q

Describe Type 1B topoisomerases

A

cut one strand of DNA
hold one strand and let the other spin around (less controlled than type 1A)
rotation is spontaneous, relieves supercoiling, doesn’t need ATP
can relieve supercoiling from either direction i.e. makes W closer to 0 from either direction
the number of rotations is not fixed so L can move by “+/-n” each time

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65
Q

Describe Type II topoisomerases

A 
cleave both strands 
double helix is passed through the gap
changes linking number by +/-2
can bring W closer to 0 from either direction 
requires ATP hydrolysis
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66
Q

Describe bacterial DNA gyrase

A

specific type of type II topoisomerase
uses ATP to introduce negative supercoiling
moves W away from 0 to make DNA more negatively supercoiled

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67
Q

What is made first NDPs or dNTPS?

A

all dNTPs are made from NTPs

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68
Q

Which nucleotides are made de novo? Which can be salvaged?

A

they are all made de novo, purines can also be salvaged

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69
Q

Explain purine synthesis

A

start with R5P
activate the 1’ carbon by adding PPi, making PRPP
add an N to to the 1’ carbon get phosphribosyl beta-1-amine
then assemble the base on the sugar (9 steps)
it is now a purine IMP (inosine monophosphate)
Note: the base is hypoxanthine, the nucleotide is IMP
now branches, one way you get ATP, the other GTP

For GTP:
XMP 
GMP (ATP to AMP)
GDP (ATP to ADP, guanylate kinase)
GTP (ATP to ADP, non-specific nucleoside diphosphate kinase) 
For ATP: 
Adenylosuccinate (GTP to GDP)
AMP 
ADP (ATP to ADP, adenylate kinase) 
ATP
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70
Q

Explain regulation of purine synthesis

A

AMP and GMP compete with IMP for their enzymes (each for their own)
ADP and GDP allosterically inhibit the step from R5P to PRPP
AMP, ADP, ATP, GMP, GDP and GTP all allosterically inhibit PRPP to phosphoribosyl-beta-1-amine

** remember these are all NTPs not dNTPs**
there isn’t anything to activate this path, its probably always on unless you need to turn it off

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71
Q

Explain pyrimidine synthesis

A 
HCO3- + glutamate + H20 
carbamoyl phosphate (2 ATP to ADP)
oroate is synthesized (add aspartate) 
add it to PRPP (assemble pyrimidine then put on sugar, lose PPi) 
this give OMP
OMP gets converted to UMP 
UDP (using ATP)
UTP (using ATP)
CTP

ATP is added to phosphorylate using the same non-specific nucleoside diphosphate kinase as purines

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72
Q

Explain pyrimidine regulation (in animals)

A

UDP and UTP allosterically inhibit the step making carbamoyl phosphate

PRPP and ATP itself will allosterically activate the reaction making carbamoyl phosphate

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73
Q

Explain purine salvaging

A

if free adenine, guanine or hypoxanthine are floating around they are joined to PRPP to make AMP, GMP or IMP

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74
Q

How can pyrimidines be reused?

A

the free bases aren’t salvaged

but if the full nucleotide is floating around they can be phosphorylated and reused

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75
Q

What does ribonucleotide reductase do?

A

it reduces any NDP to dNDP
only works at the diphosphate level, need to remove a phosphate from CTP because this is the level its made at
uses NADPH, thioredoxin and thioredxon reductase because it is a reduction reaction

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76
Q

Does TDP exist in cells?

A

NO

77
Q

Explain the regulation of ribonucleotide reductase

A

has a catalytic site, an activity site and a specificity site
has 2 of each (dimer)

when ATP is high it binds to the A site and turns the enzyme on
ATP also binds to the S sure and this cause it to favour pyrimidines
dNTP forms of CDP and UDP are made
they can be used to make dTTP
dTTP displaces ATP from the S site
dGDP is then made
dGDP builds up and binds to the S site
dADP is then made
dADP gets phosphorylated
dATP binds to the A site and shuts off the enzyme

78
Q

Explain how dTTP is made

A 
from dUDP:
dUDP
dUTP
dUMP (lose PPi) 
dTMP 
dTDP (uses ATP)
dTTP (uses ATP)
from dCDP:
dCDP
dCMP
dUMP
dTMP etc
79
Q

Why does the cell just go from dUTP to dTTP?

A

because polymerases can tell the difference between dUTP and dTTP so there is a specific enzyme that immediately removes the phosphates from dUTP

80
Q

Why go from dUDP to dUTP then back to dUMP?

A

there are non-specific kinases that probably phosphorylate it because they don’t distinguish between nucleotides

81
Q

Explain purine degradation

A 
remove the phosphate 
remove the (deoxy)sugar 
get xanthine eventually 
convert to uric acid 
same strategy is used for GMP, AMP, IMP, XMP and the deoxy forms
82
Q

Explain pyrimidine degradation from free bases

A

remove an ammonia, CO2 and an amino acid (which aa depends on which pyrimidine)
the AAs get converted to fatty acyl-CoA derivatives
get fed into fatty acid metabolism
i.e. they get broken down instead of excreted like purines

83
Q

Describe E coli’s origin of replication

A

Circulare genome, has one origin
Have an area that is AT rich and also contains several repeats
there are also 4 DnaA sites which DnaA binds to
Dam methylase methylates the A in every GATC sequence

84
Q

Explain replication initation in E coli

A

DnaA bound to ATP associates with its binding sites, polymerizes, causes the DNA to fold in a certain way and serves as a signal for HU to come in
HU is a small positively charged histone like protein that separates the DNA (is NOT a helicase)
DnaC loads DnaB (DnaB is the helicase, DnaC is its chaperone)
this is called the prepriming complex

85
Q

Describe DnaB. How is it loaded?

A

E coli helicase
homohexamer
a SINGLE strand of DNA goes through the centre
is on the lagging strand template in E coli
hydrolyses an ATP for every base that it advances
DnaC helps load it by binding so there is gap between two of the monomers

86
Q

How is bacterial invitation regulated?

A

a protein called SeqA binds to hemimethylated sites and DnaA can’t bind (at OriC)
once the DNA is fully methylated SeqA dissociates and DnaA can bind again

DnaA-ATP binds to DNA and is then hydrolyzed to DnaA-ADP (inactive)
it takes a while for the DnaA-ADP to be turned back to DnaA-ATP

87
Q

What kind of supercoiling do you generate as you unwinding DNA?

A

lots of positive writhe (because its from overwinding)

left-handed

88
Q

Explain why supercoiling is favoured in vivo

A

in bacteria DNA gyrase introduces negative supercoiling, in us our DNA is wrapped around histones
Note: it is spiral writhe, so negative is left-handed

89
Q

How are topoisomerases used during replication?

A

Type IB and type II topoisomerases help relieve positive supercoiling i.e. the natural negative supercoiling isn’t enough

90
Q

Describe bacteria’s single strand binding protein

A

called SSB
is a homotetramer
each monomer has an olignucleotide/oligosaccharide binding fold
make interactions with the deoxyribose backbone and also have Ws and Fs that they will stack with the bases too
bind in a non-sequence specific manner
their binding pattern depends on salt concentration
Note: eukaryotic version is RPA

91
Q

Describe bacterial primase

A

called DnaG
binds as a trimer
isn’t really active until it binds to helicase
makes about 12bp RNA primers (5’ to 3’)
subunits “bump” into each other and fall off
is pretty inaccurate

92
Q

Describe the E coli polymerases

A

Pol III is the replicative polymerase

Pol I is for filling in okazaki fragments

93
Q

Describe eukaryotic polymerases

A

Pol epsilon and delta are the main replicative

Pol alpha is the primase

94
Q

Describe the E coli DNA Pol III holoenzyme

A

has 3 primases interacting with helicase
there are 2 DNA polymerases, which have core alpha (DNA polymerase), epsilon (3’ to 5’ exonuclease) and theta (binds E) subunits
there are 2 beta sliding clamp subunits per polymerase (i.e. 4 total)
gamma complex is the clamp loader, has gamma and tau which are ATPases and delta and delta’ which open the clamp, chi binds SSB, psi binds chi and gamma

95
Q

Describe the E coli sliding clamp. How is it loaded?

A

homodimer that has 6-fold symmetry
prevents polymerase from dissociating
only binds double stranded DNA so it needs the primer to bind
gamma complex bound to ATP binds to the clamp
causes a gap to open between the subunits, so sDNA can go in
hydrolyzes ATP and releases clamp

96
Q

When does polymerase go its max speed?

A

when it is bound to the sliding clamp (approaches 1000bp/sec)

97
Q

Describe the catalytic domain of polymerases and how it works

A

palm has the synthesis and exonuclease sites
binding site for incoming NTPs/dNTPs is in the finger domain
finger domain changes conformation to close around the bp, this is necessary to see if its right
binds, synthesizes, then shifts everything
if there is a mismatch the daughter strand frays and enters the exonuclease site

98
Q

What is the energy source for chain elongation in DNA replication?

A

release of PPi from an incoming dNTP

99
Q

Explain a mechanism of catalysis in DNA replication

A

divalent metal ions stabilize the negative charges, there are coordinated by various side chains on the proteins
usually zinc
zinc ions also arrange the geometry perfectly, but only if you’re making the right base pair (helps prevent mismatches)

100
Q

Describe the Okazaki fragment cycle in E coli

A

clamp dissociates from one okazaki fragment and moves on top the next
clamp can’t bind again until the primer has been laid down

101
Q

Can the clamp loader bind the polymerase and clamp at the same time?

A

no

102
Q

Describe the trombone model

A

loop of the lagging strand gets bigger as helicase moves,

DNA polymerases then dissociates and it can start over

103
Q

Describe Okazaki fragment maturation in E coli

A

Pol III either bumps into the RNA primer or primase gives it a signal (would leave a small gap in this case) to dissociate
Pol I is recruited
Note: the sliding clamp doesn’t fall off it stays on
Pol I has 5’ to 3’ exonuclease activity, chews up the primer and lays down more DNA as it goes along
if the whole primer doesn’t get taken out RNase H will come in and get the rest
DNA ligase also binds to the sliding clamp, and ligates the nicks
once it is all done (whole chromosome because E coli only has one ori) there is a signal for the clamp to come off

104
Q

What can stall replication and cause one fork to move faster than the other in E coli?

A

head-on collisions with transcription machinery (if they’re going in opposite directions)

105
Q

Explain replication termination in E coli

A

there are termination sites that Tus protein binds to
Tus lets replication machinery go past in one direction, but not the other
actually coming off probably has to do with transcription machinery because once it gets to the other side, genes will be being transcribed in the opposite direction DNA is being synthesized

106
Q

Describe how eukaryotic replication initiation is different

A

there are many origins that are 3-300kbp apart
each one is only used once per cell cycle
there is no consensus sequence for them, just wherever ORC binds
not all origins start at once

107
Q

What are the 2 stages of eukaryotic replication initiation called?

A

licensing (assembly of prereplication complexes) and activation

108
Q

Describe the licensing stage in eukaryotic replication initiation (yeast)

A

happens in G1, is inactive until S phase
ORC (origin recognition complex) is a heterohexamer that binds to the origin
Cdc6 then binds
then Cdt1 binds MCM complex and helps bring it in to bind
MCM is also a heterohexamer (is the helicase), only binds ssDNA, leading strand in eukaryotes
Cdt1 is functionally similar to DnaC
this is the pre replication complex

109
Q

Describe the activation step in eukaryotic replication initiation (yeast)

A

at entry into S phase CDKs and Cdc7-Dbf4 phosphorylate helicase and other proteins
11-3-2 complex binds
this turns on the replication machinery and recruits polymerase
helicase starts unwinding DNA and replication starts
ORC stays behind, while the rest of the complex moves

110
Q

How do yeast make sure each origin is only used once per cell cycle?

A

some of the component i.e. Cdc6, once phosphorylated are degraded
this stops more complexes from forming
Note: the ones that are already in complexes aren’t degraded
there is also a protein called geminin that binds and inactivates Cdt1 in all phases except for G1 (moves it out of the nucleus and inactivates it)
complexes can only be formed when it is released

111
Q

What is the eukaryotic helicase called?

A

MCM2-7 (E coi is DnaB)

112
Q

What is the clamp loader in eukaryotes?

A

replication factor C (RFC), (gamma complex in E coli)

113
Q

What is the sliding clamp in eukaryotes?

A

PCNA (beta subunit dimer in E coli)
Note: PCNA is a homotrimer (beta is dimer)
PCNA and beta dimer don’t have any homology but have basically the same structure
Remember: only bind dsDNA

114
Q

What are the replicative polymerases in eukaryotes?

A

Pol delta binds the lagging strand
Pol epsilon binds the leading strand
(Pol III in E coli)

115
Q

What is the eukaryotic primase?

A

contains a primase, Pol alpha and an accessory protein
puts down about 10 bases of RNA and 30 bases of DNA
(E coli is DnaG)

116
Q

What is the SSB in eukaryotes?

A

replication protein A (RPA)

117
Q

Describe Okazaki fragment maturation in eukaryotes

A

replicative polymerase just continues i.e. there isn’t another one
just displaces the strand, including some of the previous okazaki fragment, may or may not get all of the DNA that was part of the primer
there is an endonuclease that recognizes the flap and cuts it off
eventually the clamp loader makes it dissociate and loads it onto the next okazaki fragment
Remember: the clamp stays behind because ligase binds to it

118
Q

Are the nucleosomes on new DNA the same as old?

A

yes
same modifications and placement
new and old are distributed evenly

119
Q

What degrades the last primer on the end of a chromosome? Are the ends of both chromosomes shortened?

A

RNase H etc
helicase sometimes falls off before its done too, so sometimes the leading strand is shortened as well
Remember: there are just 6bp repeats at the ends

120
Q

Describe telomeres

A

G-rich repeat sequence that is thousands of bp long
30-100bp 3’ overhang forms a t-loop with the complementary strand (loops around and displaces itself)
there is a t-loop at each end, so if replication is fully finished on one side the DNA is degraded a bit to give a 3’ overhang

121
Q

Describe telomerase’s structure and function

A

is a reverse transcriptase
expressed in embryos
has an RNA and protein part
RNA part is the template for DNA synthesis
once it extends the end long enough, a primer can be put down and a new okazaki fragment made

many of the domains in it are conserved, a lot of the sequences aren’t
has many stem loop structures that tell it how far it can go in making a template i.e. what 8bp etc that it can use

122
Q

Explain how retroviral sRNA genomes are replicated

A

retroviral RTs have 3 activities
they are RNA-directed DNA polymerases, use host tRNA as a primer
also have RNaseH activity, they degrade the RNA that is part of the RNA-DNA hybrid
also have DNA-directed DNA polymerase activity, so they then synthesize the other side of the DNA strand (RNaseH leaves behind little pieces of RNA that act as primers, there are ways to get rid of them later)
dsDNA is then incorporated into the host genome

123
Q

What areas of the genome are most susceptible to polymerase slippage?

A

microsatellites

124
Q

Explain Huntington’s disease

A

PolyQ repeats (CAG)
exapnsion to more than 35 repeats in hutingtin protein
causes gradual neuronal death

125
Q

Explain Fragile X syndrome

A

CGG repeats in the 5’UTR of the FMRP gene
get disease when there are more than 200 repeats
leads to silencing of the gene
has neurological and developmental problems

126
Q

What happens when cytosine gets deaminated? A? G? 5-methylcytosine?

A

turns into uracil
pairs with A instead of G

A becomes hypoxathine
G becomes xanthine

5Me-C becomes T, which is especially mutagenic because it can’t be recognized as abnormal in DNA

127
Q

What is deamination?

A

amino group gets replaced by a double bonded oxygen

128
Q

What does nitrous acid do? (HNO2)

A

deamination

129
Q

Give an example of oxidative damage using 8-oxo-G

A

8-oxo-G is very mutagenic
G can be oxidized at the 6th position
makes it go from the anti to syn position
now pairs with A

130
Q

What is alkylation? Give some examples

A

the addition of some kind of molecule from the environment
ie nitrogen mustard will react with Gs and give you a cross-link
or
benzopyrene will give an adduct by covalently bonding to G

131
Q

What can intercalating agents do?

A

cause slippage
give frameshift mutations
more difficult for polymerases to get in
ie ethidium bromide

132
Q

Explain the Ames test

A

take histidine auxotrophs
put on a plate that doesn’t have His in it
put mutagen on filter paper in the middle of the dish
it will diffuse out and you will get a gradient
will get a clear zone
will get some revertants
this shows that the chemical is increasing the mutation rate
doesn’t mean it is carcinogen, need to do animal tests

133
Q

Describe direct repair via photoreactivation

A

expose bacteria to UV light, seem dead, put them in visible light they come back to life
have an enzyme called DNA photolyase
has 2 cofactors, a chromophore that absorbs visible light and passes it to FADH
FADH can use it to rearrange electrons in pyrimidine dimers do to UV damage
essentially just reverse it

134
Q

Describe direct repair of methylated guanine

A

methylated guanine (O6-Me-G) pairs with T instead of C
enzyme called guanine methyltransferase takes off the methyl
the enzyme is then itself degraded
“suicide enzyme”
not really actually an enzyme

135
Q

What is the net effect of DNA repair systems?

A

mutation rate from 10^-6 to 10^-8

136
Q

Explain base excision repair

A

fixes single bases that don’t distort the DNA helix very much and single strand nicks
i.e. deamination of A to hypoxanthine

a specific glycosylase comes in and recognizes the damage, removes base and leaves backbone (AP site)
an AP endonuclease creates a nick in the backbone, leaving a 3’OH and 5’ deoxyribose phosphate

in E coli Pol I has 5’ to 3’ exonuclease activity so it takes it out and polymerizes new bases in it place

in eukaryotes have 2 paths from here
“long patch”
either polymerase displaces some bases and you get a flap which is then removed
or
“short patch”
Pol beta which has 5’ deoxyribosephosphate lyase activity takes out the 5’ deoxyribose phosphate and replaces it

137
Q

What is Pol beta?

A

has 5’ deoxyribosephosphate lyase activity, involved in short patch base excision repair

138
Q

How do glycosylases recognize bases they need to remove?

A

may be from proteins binding and being able to detect small distortions
but also by base flipping
ie uracil DNA glycosylase
flips bases out using a leucine that makes base stacking-like interactions with bases that were on either side
cut out dU if it finds one

139
Q

What is weird about the glycosylase used for 8-oxo-G?

A

when it finds 8-oxo-G paired with A it cuts out the A

once its paired with C there is another enzyme that will take out the 8-oxo-G

140
Q

What does mismatch repair fix?

A

single base mismatches and small loops i.e. 3-5bp

141
Q

Explain mismatch repair in E coli

A

MutS detects the damage and brings in MutL
MutS-MutL complex recruits MutH
MutH creates a single strand break at a nearby GATC site
there are some steps to orient it properly
helicase II comes in and so does an exonuclease which degrades a chunk of one strand
Pol III fills it in and ligase seals it

142
Q

Describe how MutS works in E coli

A

dimeric protein
DNA binds to it and is under stress
if there is a mismatch it is more likely to kink
if it kinks then MutL is recruited
MutS-MutL scans and finds the nearest GATC site
MutH comes in and clips the unmethylated strand
need to use 5’ to 3’ or 3’ to 5’ exonuclease depending on which direction the GATC site was

143
Q

Where does dam methylation occur in e coli? Where does the methyl group project?

A

N6 of the adenine in GATC sequences

methyl projects into the major groove and can be recognized by proteins

144
Q

In the experiment to determine the role of methylation is e coli MMR which strand had which mutation?

A

V strand had Hind III mutation (target is AAGCTT)

C strand had XhoI mutation (target is CTCGAG)

145
Q

What are the eukaryotic homologs of MutS called? MutL? MutH?

A

MutS homologs are MSH, form heterodimers (instead of homodimers)
there are different MSH proteins for different types of damage
MutL homologs are MLH, also have PMS proteins
MLH and PMS proteins also function as heterodimers and bind to MSH proteins
there is NO MutH homolog!

146
Q

How are mismatches repaired in eukaryotic MMR?

A

lagging strand
recognized the right strand because new strand has nicks in it
this is why you don’t need a MutH homolog to nick
exonuclease removes mismatch

leading strand
pol epsilon misincorporates an rNTP into DNA every 1000bp or so
it gets removed by RNaseH2
leaves a nick

147
Q

Explain MMR and hereditary nonpolyposis colorectal cancer

A

if you have mutations in MSH or MLH proteins you become more susceptible to this cancer
initial lack of one of the alleles of either MLH1 or MSH2 makes it much more likely that you will get mutations in APC, ras, then p53 eventually resulting in a metastatic carcinoma
another way to recognize it is that when MMR is defective, micro satellites aren’t stable and their lengths will differ in tumour cells compared to normal cells

148
Q

What is NER used to repair?

A

distortions involving large adducts or multiple base pairs
thymine dimers in eukaryotes
in eukaryotes it also rescues stalled RNA polymerase

149
Q

Explain NER is E coli

A

UvrA and UvrB proteins recognize the distortion and form a complex at the site
denature the DNA
exinuclease is recruited, it nicks about 6 bases from the distortion on either side
UvrD, a helicase, takes out these 12-13nt
Pol I and ligase come in and fix it

150
Q

Explain NER and xerodoma pigmentosum

A

XP gives extreme sensitivity to sunlight and susceptibility to melanoma
J Cleaver did experiments using cell extracts from patients and showed that they would not repair DNA
also showed that there were complement groups (now there are 8 genes)
these are the genes involved in our NER pathway

151
Q

What pathway does our NER repair path overlap with?

A

transcription-coupled repair

ie NER in humans also recognizes stalled RNA polymerase

152
Q

Explain eukaryotic NER

A

XPC recognizes the thymine dimer etc
binding results in denaturation of the strands
XPB and XPD bind to the strand with the lesion (RNA polymerase has XPB and XPD in it)
XPF and XPG are nucleases that come in and nick upstream and downstream of the lesion
helicase takes out 24-32 nt
polymerase and ligase come in

153
Q

Describe Fanconi Anemia

A

rare hereditary disorder
anemia, hearing failure, limb deformities and susceptibility to leukemia
caused by mutations in ICL repair paths

154
Q

Explain ICL repair in eukaryotes

A

anchor complex that contains FANCM recognizes the ICL and binds
this recruits the core complex
core complex ubiquitinates FANCI and FANCD2
which somehow activates FANCP and FANCQ, which are nucleases, as well as other proteins
results in the breakage of one strand, unhooking of the ICL and translesion synthesis
some info is lost
it appears that two replication forks need to hit each other in order to repair an ICL

155
Q

Explain how fork convergence was identified as being important for ICL repair

A

set up a plasmid with a single ICL in it
put 48 repeats of the Lac operator infront of it on one side
when the lac repressor is bound the replication fork has a hard time getting past
the ICL is not fixed
if IPTG was added, the lac repressor was released and the forks converged and the ICL was fixed

156
Q

Fanconi anemia

A

ICL repair is defective

often get acute myeloid leukemia

157
Q

XP

A

NER is defective

melanoma

158
Q

HNPCC

A

MMR is defective

colon cancer

159
Q

BRCA1 and BRCA2

A

involved somehow with repairing DSBs

get breast cancer

160
Q

What are UumC and UumD?

A

proteins that are induced by DNA damage in E coli

161
Q

Explain the experiment that was used to identify that UumC is a translation polymerase

A

used a strain that had no Pol I and had temp sensitive Pol III
even at the higher temperature the fractions containing UumC had polymerization activity

162
Q

How do translesion polymerases compare structurally to normal ones?

A

have a very reduced finger and no exonuclease
active site is much more open
allows bases to polymerize even if they aren’t paired properly

163
Q

How is translesion synthesis controlled in eukaryotes?

A

PCNA (the clamp in eukaryotes) gets ubiquitinated when there is DNA damage
eukaryotic Y family translesion polymerases have a ubiquitin binding site
bind to the clamp/DNA and synthesize past the lesion

164
Q

Explain the SOS response in E coli

A

global transcriptional response in E coli due to DNA damage
affects the transcription of more than 40 genes
is initiated by a protein called RecA that binds to ssDNA as a filament
all of the genes involved in the SOS response have a LexA operator (LexA is a transcriptional repressor)
when RecA polymerizes it catalyzes the self-cleavage of LexA
genes are able to turn on
the time and duration of de-repression depends on the location and binding affinity
RecA, BER and NER genes are turned on first
get UumCD complex later

165
Q

What is the SOS response in eukaryotes called?

A

DDR (DNA damage response)

166
Q

If damage cannot be repaired in eukaryotic cells what happens?

A

apoptosis

167
Q

What is ataxia telangiectasia?

A

caused by mutations in ATM, a transducer protein (protein kinase)
brain degeneration, radiation sensitivity, immune problems, cancer susceptibility

168
Q

How does ATM work?

A

when a DSB is sensed the MRN complex comes in and binds and somehow activates ATM
ATM will phosphorylate CHK2 which then phosphorylates CDC25A (a phosphatase)
CDC25A is degraded and cannot dephosphorylate CDK2-cyclin, so the cell cycle stops

ATM and CHK2 both also phosphorylate and stabilize p53, a transcription factor
activates transcription of p21
p21 inhibits CDK2-cyclin complexes

169
Q

Where is homologous recombination used for repair?

A

DSBs
fork stalls
gap repair

170
Q

Explain the general pathway for DSB repair by homologous recombination

A

selectively degrade the 5’ ends to give 3’ overhangs
3’ overhang scans along homologous chromosome using a recombinase to find a similar sequence
get second strand invasion
strand starts getting synthesized and you get a branched intermediate
then have two choices
1
synthesis-dependent strand annealing (SDSA)
pull apart the branched intermediate, reassociate and finish synthesizing
or
2
DSBR (occurs less often)
complete replication while the junction is still in place
get 2 holiday junctions that need to be cleaved
depending where you cleave them you either get a crossover or non-crossover event

171
Q

How are collapsed forks fixed?

A

have a nick in one strand, fork collapses
process the 5’ end to give 3’ overhang
strand invasion, there is only one strand so it creates a fork
get branch migration backwards to create a holiday junction
resolve the holiday intermediate then restart replication
basically just re-attatch the broken arm

172
Q

What is RecBCD and how does it work

A

a trimer that processes ends for recombination in E coli
RecC holds the other units together
RecB and RecD have helicase activities (requires ATP)
(one is 5’ to 3’ the other is 3’ to 5’)
RecB also has a nuclease domain
RecC binds to the chi sequence in e coli
there is an acidic pin in the middle that pulls the strands apart
3’ end gets cut at every base, 5’ end gets cut about every 5 bases
chi sequence on 3’ end binds to RecC and stops it from being degraded, this gives the 3’ overhang

173
Q

What does the chi sequence do?

A

thought to be protective, DNA without it will be degraded

presence of them increases recombination, but only on one side because they aren’t palindromic

174
Q

How is RecBCD involved with RecA?

A

RecA is the E coli recombinase

RcBCD helps exchange SSB for it on ssDNA

175
Q

Explain how RecA acts as a recombinase

A

binds ssDNA, isn’t active until it does so (conformational change), binds cooperatively 5’ to 3’ and also disassembles this way
makes a filament along it
is an ATPase
promotes strand invasion and helps search for a homologous sequence
has 2 binding sites, one for ssDNA, the other for dsDNA
probably flips base pairs to find ones that go together

176
Q

How is RecA loaded in gap repair?

A

by RecFOR because there are no free ends i.e. can’t use RecBCD

177
Q

What is RuvAB?

A

used for branch migration in E coli recombination

178
Q

What is RuvC?

A

resolvase in E coli recombination

179
Q

Explain the regulation of RecA

A

autoregulated
C-terminal tail that keeps it turned off, unless moved or mutated to be gone
by other proteins
RecA levels are responsive to LexA levels
RecBCD and RecFOR load it
RecX and UvrD mediate its removal
DinI stabilizes it

180
Q

Explain what RuvAB does

A

accelerates branch migration (compared to what RecA can do itself)
RuvA is a tetramer that binds to holiday junctions using positively charged grooves (may bind as an octamer)
RuvB is an ATPase and translocase
binds on either side of RuvA and hydrolyzes ATP and pulls DNA strands outwards causing branch migration
breaks H bonds from one set of strands, they reform with the other one i.e. no net H bonds

181
Q

What does RuvC do?

A 
resolvase in E coli 
acts as a dimer 
has some sequence specificity ATTTGC
this sequence needs to be in the holiday junction before it can cut 
binds in concert with RuvAB
182
Q

What is the XerCD system?

A

system in e coli that helps resolve fix a dimeric genome if a holiday junction isn’t cut properly

183
Q

What is special about Deinococcus radiodurans?

A

much more resistant to radiation than other organisms because it is so good at DNA repair and homologous recombination

184
Q

What is Rad51?

A

eukaryotic RecA (recombinase)

185
Q

What is Spo11?

A

protein that makes DSBs for homologous recombination in meiosis in eukaryotes

186
Q

How do yeast switch mating types?

A

homologus recombination at the MAT locus on chromosome 3 using either the HMLalpha or HMRa sequence
HO nuclease makes a sequence-specific DSB at the MAT locus
get 5’ to 3’ degradation
Rad51-mediated strand invasion
the non homologous sequence is degraded
second strand is displaced and you get synthesis

187
Q

What is another name for BRCA1?

A

FancS

note: it gets phosphorylated by ATM

188
Q

What does BRCA2 do?

A

helps load Rad51 onto ssDNA displacing RPA

189
Q

Explain non-homologous end joining in eukaryotes

A

Ku70/80 is a dimer that recognizes the DNA ends of a DSB
DNA-PKcs (a protein kinase) binds to Ku70/80, it is responsible for trying to hold the ends together
it phosphorylates itself and brings in the Artemis nuclease
Artemis widens the break (lose some info)
there is an annealing process that only needs a couple bases of homology
ligases seal everything back together

Biochem 3382 (4 decks)

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