EXAM 2 - Session 13: DNA Stucture and Replication

Question 1

Describe the structure of DNA.

Answer 1

3D structure - 2 helices wrapping around each other
* surface has major and minor grooves
* size of groove affects access to bases within helix

Question 2

What are the types of interactions that can affect DNA as drug targets?

Answer 2

strand breaker - bleomycin

non-covalent interaction
* intercalation between bases
* fitting into minor grooves, major grooves, or spanning both

covalent complexes - cis platin

Question 3

What is cis-platin used for?

Answer 3

Cis platin is an anticancer chemotherapy drug through covalent binding (involves at least one Guanine)
* Covalent bonding across helix or along one strand will block RNA and DNA polymerase –> decrease DNA replication or mRNA production of cell growth
* affects normal cells too - but affinity for rapid-metabolizing cells

Question 4

What are the different binding methods of cis-platin?

Answer 4

Bond is formed between platinum and nitrogen on a guanine and another nucleotide.
* interstrand (~3-5%)
* intrastrand (~80-90%)
* either strand (~3%)

Question 5

Explain interstrand cis platin covalent binding.

Answer 5

Interstrand - binding between consecutive nucleotides of opposite strands.

Question 6

Explain intrastrand cis platin covalent binding.

Answer 6

Intrastrand - covalent binding between consecutive or nearby nucleotides on the same strand.
* majority of cis platin bonds are between consecutive nucleotides.

Question 7

Explain either strand cis platin covalent binding.

Answer 7

Either strand - cis platin is connected to a protein on one end and a nucleotide on the other.
* only connected to one strand.

Question 8

How and why is cis platin binding detrimental to cancer cells? Hb other cells?

Answer 8

Covalent bonding across helix or along one strand blocks DNA/RNA polymerase –> decreases DNA replication or mRNA production for cell growth.
* normal (slower) growing cells are less affected by cis platin

Question 9

What is the role of topoisomerase in DNA replication?

Answer 9

Topoisomerase is reponsible for preventing the DNA double helix ahead of the replication fork from getting too tightly wound as the DNA is opening up.
* cleave DNA strands in order to provide stress relief
* untangle replicating DNA

Question 10

Describe the steps of DNA replication.

Answer 10

1. Topoisomerases cleave and rejoin the DNA strand to prevent excessive stress/tension.
2. Single-strand binding proteins stabilize the unwound parental DNA.
3. The leading strand is synthesized continuously in the 5’–>3’ direction by DNA polymerase.
4. The lagging strand is synthesized discontinuously. Primase sythesizes a short RNA primer, which is extended by DNA polymerase to form an Okazaki fragment.
5. After the RNA primer is replaced by DNA (by another DNA polymerase), DNA ligase joins the Okazaki fragment to the growing strand.

Question 11

What is the main function of RNA primer?

Answer 11

RNA primer is required to provide initial 3’ OH for DNA synthesis to start (add nucleotides)

Question 12

What direction does DNA replication go?

Answer 12

Replication proceeds from 5’ to 3’

Question 13

Describe the two kinds of daughter strands.

Answer 13

Leading strand - continuous synthesis
Lagging strand - short, discontinuous synthesis yields seperate lengths of DNA = Okazaki fragments
* RNA primers are removed by RNAseH enzyme
* DNA nucleotides fill in gaps
* ligase joins fragments together

Question 14

Describe the consequence of the 5’ –> 3’ rule.

Answer 14

The chromosome ends (telomeres) shorten with repeated rounds of DNA replication.
* After RNA primers are removed, there is a single-stranded DNA template at the chromosome terminus left
* The single-stranded DNA template that is leftover gets degraded by exonucleases.

Result: both ends of chromosome (telomeres) are shortened each time the DNA is replicated.

Question 15

Describe the role of telomeres and what happens when they are shortened too much.

Answer 15

Telomeres - ends of linear eukaryotic chromosomes
* protect and stabilize internal section of chromosome
* if telomere is shortened too much, cell stops replicating = ‘biological clock’ of cellular age (since the telomeres are so short, they can’t afford to lose more from the CODING regions of the DNA)

Question 16

Explain the End Replication Problem of DNA synthesis.

Answer 16

• DNA polymerase needs RNA primer to start replication.
• After primer is removed, single strand DNA is left.
• single strand region is removed by exonuclease and chromosome shortens.
• When the telomere is too short, the cell begins to degrade CODING regions near the telomeres (telomeres are non-coding).

Question 17

Can telomeres be rebuilt? How?

Answer 17

Telomerase - rebuilds the length of telomeres
* adds onto the 3’ end of the DNA by using telomerase
* reverse transcriptase - synthesizing DNA from RNA template
* adds nucleotides to single-stranded overhang which becomes long enough for primer to bind to and extend synthesizing strand
* when primer is removed, the single-stranded overhang is now the portion added by telomerase. (less shortening of chromosome)

Question 18

The majority of normal cells do NOT produce telomerase. What is the consequence for the tissue/organ?

Answer 18

Telomeres shorten –> cell stops dividing –> replication is halted
* tissue is worn down without new cell replacements
* If telomeres were extended –> then continued cell replication

Question 19

Where are telomerases found at high levels?

Answer 19

90% of cancer types have high telomerase levels
* levels increase from early to late stage cancer
* telomeres are maintained; cells divide and escape replicative senescence (halted replication)

Question 20

Other than telomerase activity, what other method do cancer cells use to maintain telomere length?

Answer 20

~10 % of cancer cells use alternate lengthening of telomeres (ALT) method
* sister chromosome serves as template in process similar to homologous recombination
* single telomere on one strand is the template for telomere extension on another strand.

Question 21

Explain how telomerase can act as a drug target.

Answer 21

Telomerase function blocked by “antisense” DNA
* complimentary to RNA component of telomerase enzyme
* RNA of telomerase is pre-occupied and cannot bind to the telomere
* result: telomere shortens

Question 22

Explain the mechanism of Imetalstat.

Answer 22

Imetelstat binds to template region of RNA component of telomerase –> direct, competitive inhibition of telomerase ezymatic activity
* result: suppress proliferation

Question 23

Describe the function of AZT on inhibition of telomerase.

Answer 23

Azidothymidine does not allow additional nucleotides to be added because of 5’ to 3’ rule.
* AZT binds previous nucleotide (thymidine analogue)
* azido (-N3) group in 3’ position
* phosphate group of incoming nucleotide can’t bind azido-thymidine –> synthesis stops.

Question 24

Explain the role of topoisomerases.

Answer 24

Nuclear enzymes that support DNA replication.
Dual funtionality:
* induce single-stranded breaks - DNA unwinds, helix is relaxed, ssDNA available for replication
* topoisomerase rejoins DNA ends in relaxed region

Question 25

Explain the result of topoisomerase inhibition.

Answer 25

If regulation of topoisomerase is inhibited, there is accumulation of single-stranded breaks –> cell death
* topoisomerase will only break the strands and won’ t be able to reoconnect the DNA that it broke

Question 26

How do camptothecins act against topoisomerase as a chemotherapy drug.

Answer 26

Camptothecins are cytotoxic chemotherapy drugs.
* partially inhibit topoisomerase
* drug h-bonds to topoisomerase and intercalates between base pairs
* re-ligation step is inhibited
* single-stranded breaks accumulate –> cell death