DNA Structure, Analysis, Replication and Repair

Question 1

Characteristic of Genetic Material

Answer 1

1. replication
2. storage of information
3. expression of information
4. variation by mutation

Question 2

Brief History in DNA discovery as the carrier of genetic material

Answer 2

1. Friedrich Miescher - isolated cell nuclei and derived an acidic substance, now known to contain DNA, that he called nuclein
2. Phoebus A. Levene - DNA contained approximately equal amounts of four similar molecules called nucleotides
3. Erwin Chargaff - disproved Levene’s as incorrect; demonstrated most organisms do not contain precisely equal proportions of the four nucleotides
4. Frederick Griffith - experiments with several different strains of the bacterium Diplococcus pneumoniae; transformation
5. Avery, MacLeod, McCarty - first direct experimental proof that DNA, and not protein, is the biomolecule responsible for heredity; (protease, RNAse, and DNAse)

(6. Harvey & Chase) - T2 bacteriophage

Question 3

Alternative models of DNA replication

Answer 3

1. Semi-conservative model
2. Conservative model
3. Dispersive model

Question 4

• at each point along the chromosome where replication is occurring, the strands of the helix are unwound
• if replication is bidirectional, two such forks will be present, migrating in opposite directions away from the origin

Answer 4

Replication fork

Question 5

• refers to the length of DNA that is replicated following one initiation event

Answer 5

Replicon

Question 6

Leading strand: Synthesized continuously in the same direction as the replication fork.

Lagging strand: Synthesized in small fragments called Okazaki fragments in the opposite direction of the replication fork.

Question 7

four essential components required for DNA synthesis:

Answer 7

1. dNTPs: dATP, dTTP, dGTP, dCTP (deoxyribonucleoside 5’-triphosphates)(sugar-base + 3 phosphates)
2. DNA template
3. DNA polymerase (Kornberg enzyme)
4. Mg 2+ (optimizes DNA polymerase activity)

Question 8

ability to remove nucleotides from the 3’ end of the chain

Answer 8

3’ to 5’ exonuclease activity

Question 9

Five common DNA polymerases from mammals:

Answer 9

Polymerase  (alpha):
* nuclear, DNA replication, no proofreading
Polymerase  (beta):
* nuclear, DNA repair, no proofreading
Polymerase  (gamma):
* mitochondria, DNA repl., proofreading
Polymerase  (delta):
* nuclear, DNA replication, proofreading
Polymerase  (epsilon):
* nuclear, DNA repair (?), proofreading

Question 10

Segments of single-stranded DNA are called
_________

Answer 10

template strands

Question 11

_______ (a type of topoisomerase) relaxes the supercoiled DNA; relieves torsional stress

Answer 11

Gyrase

Question 12

_______ and _______ binds to the DNA at the replication fork and untwist the DNA using energy derived from ATP (adenosine triphosphate).

Answer 12

Initiator proteins
DNA helicase

Question 13

DNA primase next binds to helicase producing a complex called a ______(primase is required for synthesis)

Answer 13

primosome

Question 14

Primase synthesizes a short RNA primer of 10-12 nucleotides, to which DNA polymerase III adds nucleotides.

Question 15

The RNA primer is removed and replaced with DNA by polymerase I, and the gap is sealed with ______

Answer 15

DNA ligase

Question 16

_______ stabilize the single-stranded template DNA during the repliction process.

Answer 16

Single-stranded DNA-binding (SSB) proteins (>200)

Question 17

Model of Replication (in E. coli)

1. Initiator proteins bind to replication origin
2. DNA helicase binds to replicator proteins
3. Helicase loads onto DNA
4. Helicase denatures helix and binds with DNA primase to form primosome
5. Primase synthesizes RNA primer, which is extended as DNA chain by DNA Polymerase

Question 18

an enzyme that joins DNA strands together by forming a phosphodiester bond between adjacent nucleotides; does not add a nucleotide

Answer 18

ligase

Question 19

Each eukaryotic chromosome is one linear DNA double helix

Average ~10^8 base pairs long

With a replication rate of 2 kb/minute, replicating one human chromosome would require ~35 days.

Solution —> DNA replication initiates at many different sites simultaneously

Question 20

binds to the terminal telomere repeat and catalyzes the addition of new repeats.

Answer 20

Telomerase

Question 21

Most DNA repair occurs in the ___ phase of the eukaryotic cell cycle

Answer 21

G1 phase

Question 22

Mismatch repair occurs in the ___ phase to correct replication errors

Answer 22

G2

Question 23

encodes a protein that prevents a cell with damaged DNA from entering the S phase

Answer 23

p53 gene

Question 24

a rare, inherited disorder that increases the risk of developing many types of cancer, especially at a young age

Answer 24

Li-Fraumeni syndrome

Question 25

Maxam-Gilbert Method
Sanger Method

Question 26

Meselson-Stahl experiment = ^15N-containing medium in ^14N medium

Taylor-Woods-Hughes Experiment - labeled chromatids

Question 27

only a single region, called _____ , where replication is initiated

Answer 27

oriC

Question 28

may be achieved on both the leading and lagging strands at a single replication fork

lagging template strand is “looped” in order to invert the physical direction of synthesis, but not the biochemical direction

enzyme functions as a dimer, with each core enzyme achieving synthesis on one or the other strand

Answer 28

Concurrent DNA Synthesis

Question 29

DNA ligase seals the gaps between Okazaki fragments with a phosphodiester bond

Question 30

a ligase seals the loose ends, creating hybrid duplexes called ______, held together by a cross-bridge structure [recombination process]

Answer 30

heteroduplex DNA molecules

Question 31

position of this cross bridge can then move down the chromosome by a process referred to as ______

Answer 31

branch migration

Question 32

Process of recombination:

Answer 32

a. two paired DNA duplexes, or homologs
b. endonuclease nicking
c. strand displacement
d. ligation, creating heteroduplex DNA molecules
e. branch migration
f. duplex separation
g. 180 degree rotation; x (chi) form or Holliday structure
h & i. if the two strands on opposite homologs are now nicked by an endonuclease ligation occurs; two recombinant duplexes are created

Question 33

How PCR Works

Answer 33

Initial melt - 2 mins - 94C
Melt - 30 secs- 94C
Anneal - 30 secs - 55C
Extend - 1 min - 72C
Final extenson - 6 mins - 72C
Hold - 4C

Question 33

PCR processe (ONE Cycle)

Answer 33

1. Denaturing (95C)
2. Annealing (55C)
3. Extension (72C)

Question 34

Three main features of the DNA
synthesis reaction:

Answer 34

1. DNA polymerase I catalyzes formation of phosphodiester bond between 3’-OH of the deoxyribose (on the last nucleotide) and the 5’-phosphate of the dNTP.
   • Energy for this reaction is derived from the release of two of the three phosphates of the dNTP.
2. DNA polymerase “finds” the correct complementary dNTP at each step in the lengthening process.
   • rate ≤ 800 dNTPs/second
   • low error rate
3. Direction of synthesis is 5’ to 3’

Question 35

•Without proofreading error rate (mutation rate) is _______
•With proofreading error rate is _____ (1000-fold decrease)

Answer 35

1 x 10^-6
1 x 10^-9

Question 36

DNA replication is continuous on the
______ strand and semidiscontinuous on
the _____ strand

Answer 36

leading
lagging

Question 37

DNA replication in eukaryotes:
* Copying each eukaryotic chromosome during the S phase of the cell cycle presents some challenges:
Major checkpoints in the system:

Answer 37

• Cells must be large enough, and the environment favorable.
• Cell will not enter the mitotic phase unless all the DNA has replicated.
• Chromosomes also must be attached to the mitotic spindle for mitosis to complete.
• Checkpoints in the system include proteins call cyclins and enzymes called cyclin-dependent kinases (Cdks).

Question 38

Enzymes that synthesize nucleic acids by
forming phosphodiester(PDE) bonds

Answer 38

Polymerases

Question 39

Enzymes that hydrolyze PDE bonds

Answer 39

Nucleases

Question 40

removes nucleotides from either the5’
or the 3’ end of a nucleic acid.

Answer 40

Exonucleases

Question 41

cut within the nucleic acid and release
nucleic acid fragment

Answer 41

Endonucleases

Question 42

substrates for DNA synthesis are
the dNTPs, whereas the substrates
for RNA synthesis are the NTPs.

Question 43

STEPS OF DNA REPLICATION

Answer 43

1. The base sequence at the origin of replication is recognized and bound by the dnaA protein.
2. The two parental strands of DNA are pulled apart to form a “replication bubble “
3. Helicase uses energy from ATP to break the hydrogen bonds holding the base pairs together
4. Single-stranded DNA binding protein (SSB) binds to the single-stranded portion of each DNA strand, preventing the strands from reassociating and protecting them from degradation bynucleases.
5. Primase synthesizes a short (about 10 nucleotides) RNA primer in the 5’ ~3’ direction, beginning at the origin on each parental strand.
6. DNA polymerase III begins synthesizing DNA in the 5’ ~3’ direction, beginning at the 3‘ end of each RNA primer.
7. RNA primers are removed by DNA polymerase I.
8. Both DNA polymerase I and III have the ability to “proofread” their work by means of a 3’ ~5’ exonuclease activity.
   8.DNA ligase seals the “nicks” between Okazaki fragments, converting them to a continuous strand of DNA.
9. DNA gyrase (DNA topoisomerase II) provides a “swivel” in front of each replication fork.
   ✓ DNA gyrase inserts negative supercoils by nicking both strands of DNA, passing the DNA strands through the nick, and then resealing both strands again.

Question 44

can relieve supercoiling in DNA molecules by the transient breaking and resealing of just one of the strands of DNA.

Answer 44

DNA topoisomerase I

Question 45

are a family of drugs that block the action of topoisomerases.

Answer 45

Quinolones

Question 46

kills bacteria by inhibiting DNA gyrase

Answer 46

Nalidixic acid

Question 47

Inhibitors of eukaryotic topoisomerase II
(etoposide, teniposide) are becoming
useful as anticancer agents.

Question 48

• Replication is completed when the two replication forks meet each other on the side of the circle opposite the origin.

Question 49

are repetitive sequences at the ends of linear
DNA molecules in eukaryotic chromosomes

Answer 49

Telomeres

Question 50

Cancer cells often have relatively high levels of
telomerase, preventing the telomeres from
becoming shortened and contributing to the
immortality of malignant cells.

Question 51

• autosomal recessive disorder,
  characterized by extreme sensitivity
  to sunlight, skin freckling and
  ulcerations, and skin cancer.
• most common deficiency occurs in
  the excinuclease enzyme

Answer 51

Xeroderma pigmentosum

Question 52

• deficiency in the ability to repair
  mismatched base pairs in DNA that
  are accidentally introduced during
  replication

Answer 52

Hereditary nonpolyposis colorectal cancer

Question 53

• encodes a kinase essential for p53
  activity
• inactivated in ataxia telangiectasia
• characterized by hypersensitivity to
  x-rays and predisposition to
  lymphomas

Answer 53

ATM gene

Question 54

• refers to the length of DNA that is
  replicated following one initiation event

Answer 54

Replicon

Question 55

Three main features of the DNA
synthesis reaction:

Answer 55

1. DNA polymerase I catalyzes formation of
   phosphodiester bond between 3’-OH of the
   deoxyribose (on the last nucleotide) and the 5’-
   phosphate of the dNTP.
2. DNA polymerase “finds” the correct
   complementary dNTP at each step in the
   lengthening process.
3. Direction of synthesis is 5’ to 3’