BC7 Old exams

Question 1

1. Sketch the eukaryotic replication fork, drawing the approximate location and geometry of DNA and the approximate location of the DNA polymerases, primase clamp loader, clamp and RPA. (4 points)

Answer 1

Need to find good diagram for this

Question 2

1. Which of the following statements is true or false? (3 points)
   
   1. Pol eta is a high fidelity DNA polymerase that is both a replicative polymerase and can read over base crosslinks.
   2. RNA ubiquitination is an important functional switch to attract translesion bypass polymerases.
   3. Mitochondria have a specialized DNA polymerase and do not rely on the nuclear DNA polymerases.

Answer 2

1. False
   
   1. Pol eta is not a replicative polymerase
2. False
   
   1. during translesion synthesis, the sliding clamp is ubiquitylated
3. True
   
   1. mitochondria have DNA pol gamma

Question 3

Genetic inactivation of RNaseH2, a Rnase that can be found in the nucleus and nicks DNA containing ribonucleotides (at the ribonucleotide containing strand) was found to reduce the proper repair of DNA mismatches after DNA replication in yeast. Explain why! (3 points).

Answer 3

1. DNA polymerases occasionally incorporate ribonucleotides in the nascent strand (1)
2. Mismatch repair uses nicks to discriminate the newly synthesized strand from the templating strand (1).
3. Nicking by Rnase-H2 in the newly synthesized strand generates the strand discrimination signal and proper loading of the helicase/exonuclease for repair (1).

Question 4

TDG is a human DNA glycosylase that specifically recognizes T-G mismatches and excises the T base. Why is this enzyme important in human cells along with the canonical mismatch repair machinery? (hint: consider CpG islands) (3 points)

Answer 4

1. CpG islands are often methylated at position 5 of the cytosine 1 (1).
2. Deamination results in T:G mismatches (1).
3. These are properly repaired by TDG, but not necessarily by the mismatch repair machinery (1).

Question 5

1. Briefly explain why inactivation of uracil-N-glycosylase - an enzyme that cleaves deoxy-uracil in DNA - leads to defects in the maturation of antibodies. (2 points)

Answer 5

1. AID generates uracil by deaminating cytosine in immunoglobuline genes in activated B-cells (1).
2. UNG cleaves the uracils DNA to generate abasic sites, these are used for an error prone repair (1).

Question 6

You assembled a nucleosomal array in vitro. (3 points)

1. Which enzyme and substrate can you use to methylate H3K36?
2. How can you check if your in vitro reaction worked?
3. What do you expect to see if you bind HP1 protein to your array?

Answer 6

1. H3K36 methyltransferase (Set2)/SAM
2. Western blot with H3K36me antibody or MS
3. It doesn’t bind

Question 7

1. Which of the following statement/s is/are true? (point deduction for wrong answers!) In nucleosome assembly… (3 points)
   
   1. CAF1 deposits acetylated H3/H4 tetramer onto DNA
   2. CAF1 deposits methylated H3/H4 tetramer onto DNA
   3. NASP deposits H3/H4 tetramer onto DNA
   4. H3.3 variant is found at silent chromatin
   5. H2A.Z is enriched at the termination site
   6. Histone chaperone DAXX binds H3.3

Answer 7

1. true
2. false
3. false
4. false
5. false
6. true

Question 8

1. If you want to test whether H3K9 methylation is epigenetically inherited in vivo, (3 points)
   
   1. How would you design the experiment?
   2. Is H3K9me epigenetically inherited in wild type fission yeast cells?
   3. What do you need at the centromeric repeats in fission yeast to establish heterochromatin?

Answer 8

1. Tether H3K9methyltransferase to euchromatic DNA sequence, check if H3K9me is established, release the enzyme, check if H3K9me is maintained (2)
2. No/to some extent (0.5)
3. siRNAs (0.5)

Question 9

1. Which of the following statements is true? (3 points) (BC7 – lecture 2, BC7 – lecture 4/5)
   
   1. Chp1 chromodomain binds nucleosome core (+0.5)
   2. H3K79 methylation is deposited by Dot1 (+0.5)
   3. Ccr4-Not complex deadenylates RNA (+0.5)
   4. TFIIS promotes RNA cleavage (+0.5)
   5. Mediator interacts with activators and repressors (+0.5)
   6. H2Bub is found in active chromatin (+0.5)

Answer 9

1. true
2. true
3. true
4. true
5. true
6. true

Note: all are true

Question 10

1. You would like to determine the RNA Polymerase II localization at your gene of interest. (3 points)
   
   1. Which method and antibody against which RNA Polymerase II can you use to determine RNA Polymerase localization at the transcription initiation site?
   2. What do you expect to see at the promotor regarding the RNA Pol II amount?
   3. Which histone modification and RNA processing factors are recruited at the promotor region?

Answer 10

1. Chip with S5P CTD antibody
2. Pol2 peak
3. H3K4me3, capping factors

Question 11

Pol III, Pol δ, RNA Pol II: Function in the cell

Answer 11

1. Poll III
   
   1. If this is DNA Pol III in E. coli (is probably this one)
      
      1. Leading and Lagging strand synthesis in DNA replication
   2. If this is RNA Pol III in eukaryotes
      
      1. tRNA
      2. rRNA 5S
      3. other small RNAs found in nucleus and cytosol
2. Pol δ:
   
   1. In humans
   2. Leading strand synthesis in replication
   3. Also lagging strand synthesis in replication
3. RNA Pol II
   
   1. Transcribes: mRNA, most snRNA, microRNA

Question 12

Xeroderma pigmentosa variant is caused by a mutation in POLH a homolog of Pol eta: Why do these patients have an increased skin cancer rate?

Answer 12

1. Patients with XP phenotype often have Defects in nucleotide-excision repair genes Defects in translesion synthesis
   
   1. Pol eta is main polymerase dealing with UV crosslinks in translesion synthesis repair
   2. UV crosslinking is something that would be a problem leading to skin cancer due to the exposure of skin to UV radiation
   3. Without the machinery to fix these crosslinks, there would very likely be an increased skin cancer rate amongst these patients

Question 13

1. Xeroderma pigmentosa: Reason, involved repair pathway; What do you understand under “complementation groups”. Complementation group A contains a mutation in XPC, group B in XPA. Do you expect, that the DNA repair pathway works, if you combine cell lysates from both complementation groups? Why?

Answer 13

1. Group A has non-functioning XPC, but presumably functional XPA
2. Group B has non-functioning XPA, but presumably functional XPC
3. Since the proteins are coded from different genes, presumably combining the cell lysates would show that the genes are in fact complimentary, and the repair pathway would work again, since there would be working copies of both XPA and XPC, providing all factors needed for the repair pathway.

Question 14

1. E.coli OriC: What is the function of 9mer, 13mer and GATC for DNA replication?

Answer 14

1. 9mer: spacing, orientation and sequence
2. 13mer: A/T richness
3. GATC: Recognition site for dam methylation system

Question 15

Antibodies: How is the diversity generated by V(D)J-recombination, activation-induced deaminase (AID) and Uracil?

Answer 15

1. V(D)J-recombination
   
   1. Rag1/2 endonuclease assists in cutting at RSS site flanking V,D, or J coding segments on gene coding for antibody
   2. NHEJ repairs segment, but the targeting region is changed, with the additional help of TdT which incorporates N nucleotides
   3. This means that the antigen binding region is highly variable, allowing it to potentially bind to antigens that the organism has never before encountered.
   4. Helpful for pathogens that frequently change or ones that are completely new.
2. Activation induced deaminase (AID)
   
   1. AID induces class switch DNA recombination (CSR)
   2. Deaminases deoxycytosines in transcribed switch (S) regions
   3. Result is deoxyuracils which are removed by UNG
   4. The abasic sites are excised, resulting in SSBs that result in DSBs
   5. DSBs are repair by c-NHEJ or A-EJ (alternative end joining), leading to formation of S-S junctions and class switching
   6. Rearranges DNA that encodes heavy chain variable region, leading to antibody diversity
3. Uracil
   
   1. The change of deoxycytosine to deoxyuracil during CSR by AID leads to the rearrangements and diversity mentioned above

Question 16

1. You want to protect an in vitro transcribed mRNA from degradation by Xrn. How do you do this? Enzymes?

Answer 16

1. XRN1 is a 5’->3’ exonuclease
2. Can only degrade decapped 5’ end of mRNA, so mRNA needs to be capped to protect it
3. 5’ cap added in 3 step process, with 3 capping enzymes
   
   1. RNA triphosphatase (RTPase)
      
      1. Cet1 in yeast
      2. Cleaves 5’ terminal γ-β phosphoanhydride bond of nascent mRNA molecules
      3. Enables addition of 5’ cap
   2. Guanyltransferase (GTase)
      
      1. Yeast: eg1
      2. Adds a backwards GMP group from GTP
   3. Guanine-N7-methyltransferase
      
      1. Yeast: Abd1
      2. Methylates guanine to form final 5’ cap structure
      3. Positions RNA cap and AdoMet cofactor

Question 17

Nucleosome: What does it contain? Properties?

Answer 17

1. Contains:
   
   1. Histone core – octamer of proteins
      
      1. H2A, H2B, H3, and H4 (2 of each)
   2. Linker histones
      
      1. H1 and H5
   3. DNA
2. Properties:
   
   1. Is DNA wrapped around histone core
   2. Chromatin consists of nucleosomes strung together (beads on a string model)
   3. DNA can wrap more or less tightly in response to histone modifications or DNA modifications
   4. Keeps DNA organized and protects from damage
   5. How tightly the DNA is wrapped around histone core in nucleosome will also allow transcription of DNA or not.

Question 18

Name four, nucleosome remodelers; Properties of the +1 Nucleosome

Answer 18

Note: not sure about what is wanted with this one, do I need more specific factors, or is this enough? Also, did I say enough about the +1 nucleosome

1. INO80 family
   
   1. Incorporation and removal of histone variants – H2A.Z or H2A.X
   2. Swr1 is in family – incorporates H2A.Z at DNA damage sites
2. CDH1
   
   1. Assembly and deposition of H3.3
   2. NuRD complex is part of family, is often misregulated in many cancers
3. SWI/SNF
   
   1. Removes ectopically deposited CENP-A
4. ISWI
   
   1. Remodeling activity antagonized by H3K16 ac
5. +1 nucleosome
   
   1. Positioned at downstream boundary of nucleosome free region
   2. Often strongly positioned H2A.Z variants

Question 19

1. Which statements are correct:
   
   1. Black chromatin is deacetylated
   2. Ccr4/Not degrades polyadenine-tail of mRNAs
   3. S2P is involved in transcription termination
   4. H2Bub forms Euchromatin (?)
   5. Transcription is regulated by a mediator binding a repressor/activator

Answer 19

1. Black chromatin is deacetylated
   
   1. true
2. Ccr4/Not degrades polyadenine-tail of mRNAs
   
   1. true
3. S2P is involved in transcription termination
   
   1. True
   2. 3’-mRNA processing and termination
4. H2Bub forms Euchromatin (?)
   
   1. True?
   2. This is a sort of weird question, but H2Bub prevents compaction, so I suppose you could say that it helps form euchromatin –
5. Transcription is regulated by a mediator binding a repressor/activator
   
   1. True (I’m pretty sure)
   2. Repressor or activator bind mediator in regulating transcription

Question 20

How many copies of H2A/H2B/H3/H4 has the cell to synthesize during replication of a diploid cell? Assume that the haploid genome contains 3x10⁹ bp DNA, the linker between the nucleosomes contains 50 bp DNA and that all parental histones are maintained.

Answer 20

1. One NCP contains 2 copies of each Histone
2. ~146 bp DNA wrapped around core + 50 bp for linker = ~196 bp/octomer
3. Haploid has two copies per chromosome, so divide 3x10⁹ by 2 = 1.5x10⁹ bp
4. (1.5x10^9 / 196) = # of NCP octamers
5. # of octamers *2 = number of copies of each histone

Not sure about this one, we don’t have a calculator, so I don’t know if there was supposed to be an easier answer or something

Question 21

1. What are the 3 functions of the eukaryotic general transcription factor TFIIH?

Answer 21

1. DNA melting
2. CTD kinase
3. Transcription coupled DNA repair

Question 22

1. How are cis and trans regulatory factors/elements defined in gene regulation?

Answer 22

1. Cis = sites in DNA with specific sequence that influences transcription levels
   
   1. Examples:
      
      1. Proximal regulation elements
      2. LCR (locus control region)
      3. Enhancers
2. Trans = proteins that bind to cist sites and change the transcription levels
   
   1. Examples:
      
      1. Activators
      2. repressors

Question 23

1. What are the three key properties of the transcription elongation complex?

Answer 23

1. Very stable, which is major cause of processivity
2. Has high fidelity
3. Fast elongation rate

Question 24

1. Which two contributions can be used to quantify the strength of a bacterial transcription promotor? What is the BRE element in eukaryotic promotors?

Answer 24

1. Two contributions
   
   1. How close these 2 elements are to their respective consensus sequences
   2. The -10 element - TATAAT
   3. The -35 element - TTGACA
2. BRE is TFIIB recognition element

Question 25

1. Which ribozyme is involved in processing of pre-tRNA in bacteria?

Answer 25

1. In bacteria, pre-tRNA introns are cut out via self-splicing