Download Deamination of 5-methylcytosine yields thymine

1
Biochemistry 302, February 11, 2004
Exam 1 (100 points)
Name ____________________
I. Structural recognition (very short answer, 2 points each)
1. What form of DNA is shown on this Nature Genetics cover? Z-DNA or left-handed DNA
2. Name the bases (full names, not just single letters) that make-up this mismatched base-pair.
guanine
thymine
3. This modified base is typically found in CpG islands in eukaryotes. What is it?
5-methylcytosine
4. During what molecular process would one expect to find DNA in this configuration?
DNA recombination
2
II. Short Answer (3 points each)
1. Why is radiolabeled thymidine the best substrate to use in experiments designed to determine
the rate of cell proliferation in vitro? Thymidine will be incorporated into replicating
DNA but not RNA.
2. Provide a biochemical rationale for why AT-rich sequences are commonly found in zones of
initiation of DNA replication and near sites of transcriptional termination. Only two
hydrogen bonds needed for base-pairing between adenine and thymine versus three for
guanine and cytosine. As such, AT or AU-rich sequences are more susceptible to strand
separation or melting.
3. What is the difference between an exonuclease and an endonuclease. An endonuclease
cleaves phosphodiester bonds at sites within a polynucleotide chain while exonucleases
remove nucleotides from either the 5’ or 3’ end.
4. How is the active site of RNA polymerase biochemically converted to that of a ribonuclease?
Binding of accessory factors (such as GreA, GreB, TFIIS) donate additional amino acid
residues and a metal ion to the active site.
5. List the type of DNA damage created by cisplatinin and the repair system(s) that are able to
fix such lesions (complete spelling please, no abbreviations). Inter- and intrastrand
crosslinks via guanine bases, nucleotide excision repair or recombinatorial repair.
III. T/F (2 points each)
1. __F__ The ability of DNA to adopt a Z-form configuration is completely independent of
nucleotide sequence.
2. __F__ DNA is positively charged at pH 7.
3. __T__ 3’-deoxyadenosine is an inhibitor of RNA synthesis.
4. __F__ Acetylation of histone proteins enhances their DNA-binding affinity.
5. __F__ RNA primer excision during DNA replication is catalyzed by the ε subunit of the DNA
polymerase III holoenzyme.
6. __F__ Loading of the DNA polymerase III sliding clamp (β subunit) onto the lagging strand
need only occur ONCE during DNA replication.
7. __T__ Chemical agents that chelate divalent metal ions inhibit the polynucleotide synthetic
activities of DNA and RNA polymerases.
3
IV. Multiple choice (3 points each):
1. __A__ TFIIIA is a general transcription factor responsible for promoter recognition and
transcriptional initiation of genes encoding:
A.
B.
C.
D.
E.
5S RNA
tRNA
45S RNA
All of the above
A and B only
2. __F__ Conversion of uridylate to thymidylate requires:
A.
B.
C.
D.
E.
F.
Ribosylation
Methylation
Reduction
Oxidation
Both A and D
Both B and C
3. __B__ The principal thermodynamic parameter that accounts for the overall stability of the
DNA double helix is:
A.
B.
C.
D.
E.
Entropy
Enthalpy
Temperature
pH
Ionic strength
4. __D__ Core histone proteins comprising the nucleosome possess which of the following
biochemical properties:
A.
B.
C.
D.
E.
High arginine content
Dimerization ability
Substrate for acetylation
All of the above
None of the above
5. __A __ Nitrate-induced deamination of cytosine produces which of the following mutagenic
bases in DNA?
A.
B.
C.
D.
Uracil
Thymine
Hypoxanthine
5-azacytosine
4
6. __B__ The following activity is absent in all core eukaryotic DNA polymerases:
A.
B.
C.
D.
3’-exonuclease
5’-exonuclease
leading strand polynucleotide synthesis
lagging strand polynucleotide synthesis
7. __A__ Which of the following proteins is principally responsible for stabilization of template
strands in a non-base-paired conformation during DNA replication?
A.
B.
C.
D.
SSB
DNA Helicase
Primase
Topoisomerase
8. __C__ One of the activities below is NOT required for base excision repair.
A.
B.
C.
D.
E.
DNA N-glycosylase
Abasic endonuclease
DNA helicase
DNA polymerase
DNA ligase
9. __C__ The first line of defense in correction of a mismatched base-pair that is formed during
the course of DNA replication in E. coli is:
A.
B.
C.
D.
Base excision repair
Nucleotide excision repair
3’-exonuclease activity of DNA polymerase I
5’-exonuclease activity of DNA polymerase I
10. __A__ DNA would be predicted to be in an A-form configuration under conditions of:
A.
B.
C.
D.
Low water content
Absence of protein
High temperature
None of the above
11. __D__ RecA exhibits all of the following activities EXCEPT:
A.
B.
C.
D.
Single-stranded DNA-binding affinity
Strand exchange
ATPase
Helicase
5
12. __B__ Which of the following enzymes is inhibited by low concentrations of α-amanitin?
A. RNA polymerase I
B. RNA polymerase II
C. RNA polymerase III
13.__A__ DNA polymerase, DNA ligase, and RNA polymerase are related because each
enzyme:
A.
B.
C.
D.
Catalyzes 3’-5’ phosphodiester bond formation.
Uses nucleoside triphosphate as substrates.
Is expressed at a limiting concentration in cells.
Requires two metal ions at the active site.
14. __D__ TATA-binding protein (TBP) is best described as a:
A.
B.
C.
D.
E.
DNA-bending protein
Initiation factor for eukaryotic transcription
Recruitment factor for other components of the general transcription machinery
All of the above
None of the above
15. __A__ Mediator proteins such as Rad54 assist Rad51 (the eukaryotic homolog of RecA) in
initiating homologous recombination/strand exchange by:
A.
B.
C.
D.
E.
Enhancing recruitment of Rad51 to single-stranded DNA
Sequestering RPA (SSB) via protein-protein interaction
Activating the ATPase activity of Rad51
All of the above
None of the above
16. __B__ Depurination of DNA is induced by:
A.
B.
C.
D.
High pH
Low pH
High ionic strength
Low ionic strength
17. __B__ Mismatch repair in E. coli is dependent on the following DNA modification:
A.
B.
C.
D.
Purine alkylation
Adenine methylation
Guanine oxidation
Depyrimidination
6
V. Structural biology (12 points)
The proteins, 8-oxo-guanine DNA-N-glycosylase (OGG) and integration host factor (IHF), are
related by their ability to distort the structure of DNA but each protein does so in a different way.
Briefly describe/list 1) the biological function of each protein 2) the type of DNA alteration
induced or stabilized by each protein, and 3) how each protein mediates the structural change in
DNA at the molecular level (I’m looking for a biochemical explanation here).
OGG
1) Base excision repair enzyme or removes 8-oxoG from DNA by glycosidic bond cleavage
2) Flipping 8-oxoG out of the DNA helix (to facilitate catalysis)
3) Replacement of flipped 8-oxoG with an arginine residue to hydrogen bond with the
estranged cytosine, van der Waals forces and H-bonding between 8-oxoG and other active
site amino acids keep 8-oxoG in its “flipped out” state (in position for catalytic attack)
IHF
1) initiation factor in DNA replication and/or site-specific recombination
2) site-specific DNA-bending
3) disruption of base-stacking by intercalation of amino acid residues (proline) between
certain base-pairs in the DNA minor groove