Lecture 4: Nucleic Acids

Question 0

Describe the structure of RNA

Answer 0

RNA nucleotide is composed of a phosphate group, 5-carbon ribose sugar and one of 4 bases (uracil instead of thymine)
Single stranded polynucleotide
Nucleotides linked by phosphodiester bonds

Question 1

Describe the structure of DNA.

Answer 1

Subunit of DNA = nucleotide, which contains:
- a 5-carbon sugar (deoxyribose)
- a phosphate group
- one of 4 bases (adenine, thymine, guanine, cytosine)
Deoxyribose sugar has H on 2nd carbon
5’-3’ orientation
Sugar and phosphate of the DNA backbone are linked by strong covalent bonds (phosphodiester bonds) formed by dehydration synthesis.
Weak hydrogen bonds bind pairs of nitrogenous bases between strands of DNA
The strands of the DNA helix run in opposite directions

Question 2

Describe the process of DNA replication.

Answer 2

1. DNA helicase unwinds a segment of DNA and breaks the hydrogen bonds between the 2 strands, forming a replication fork.
2. DNA polymerase adds complimentary nucleotides to the exposed strands in the 3’ to 5’ direction, starting at the origin of replication. There are multiple origins of replication on each eukaryotic chromosome. Synthesis along the leading strand is continuous.
   Because DNA polymerase can only add new nucleotides to a free 3’ OH group, synthesis of the lagging strand is more complex.
   RNA primase binds to the lagging strand and synthesises a short RNA primer which provides the 3’ OH starting point.
   DNA polymerase III adds DNA nucleotides to the 3’ end of the RNA primer up until the next RNA primer.
   DNA polymerase 1 replaces DNA polymerase 3, removes the RNA primer and replaces is with DNA.
   DNA lipase forms a phosphodiester bond between the 3’ OH of the growing strand and the 5’ phosphate in front of it.

Question 3

Describe the three types of anti cancer drugs.

Answer 3

CROSS-LINKING AGENTS
Prevent the separation of the two DNA strands by linking them. May be intrastrand linking, interstrand linking,or linking between DNA and protein.
Includes alkylating agents (cyclophosphamide) and platinum compounds (cisplatin).
ANTIMETABOLITES
Depletes pool of nucleotides available for DNA synthesis and/or misincorporations.
Includes frolic acid antagonists (block formation of purines, eg methotrexate), purine antagonists (mercaptopurine) and pyramiding antagonists (fluoracil).
TOPOISOMERASE INHIBITORS
prevent coiling and uncoiling of DNA (eg irinotecan: prevents re-ligation of DNA, and collides with the replication complex leading to breakage of DNA.

Question 4

Describe the origin of replication.

Answer 4

DNA helicase opens up the DNA double helix at origins of replication (requires ATP).
ssDNA binding proteins stabilise DNA into a linear structure.
ssDNA binding proteins avoid the formation of hairpins, and reformation of dsDNA.
Unpaired Bases are exposed so DNA polymerase can synthesise efficiently.

Question 5

Describe DNA polymerases.

Answer 5

Synthesise new DNA strand in the 5’-3’ direction.
A sliding clamp helps DNA polymerase bind to DNA strand and improves efficiency (20 residues synthesised by DNA polymerase without clamp, 500 000 residues synthesised with clamp)
DNA POLYMERASE 3
DNA synthesis by DNA polymerase III requires template DNA, primer DNA or RNA, free nucleotides.
The cleavage of phosphate bonds provides the energy for creation of phosphodiester bonds.

Question 6

Describe DNA primase.

Answer 6

Synthesises short RNA primers required for action of DNA polymerase.
Over primer is required for leading strand, and several are required for the lagging strand.
DNA primase stops after it has made a short polynucleotide. DNA polymerase acts from the 3’ end of the RNA primer.
RNA primer is used because DNA primase has low fidelity. Once the primer is removed, any mistakes in the complimentary strand can be fixed.

Question 7

Describe Topoisomerase I.

Answer 7

Topoisomerase 1 is bound to tyrosine.
Enzyme-tyrosine complex Attaches to a DNA phosphate group and breaks a phosphodiester linkage in one strand.
This allows rotation of the two ends of the DNA double helix to relieve accumulated tension.
Energy from the breakage of the phosphodiester bond is stored in the phospho-tyrosine linkage (binds tyrosine to DNA) making the reaction reversible.
Spontaneous reformation of the phosphodiester bond regenerates both the DNA helix and the DNA topoisomerase.

Question 8

Describe telomeres.

Answer 8

Short DNA repeat sequences at end of chromosomes
Telomerase adds DNA sequence repeats to 3’ end of chromosomal DNA strands, preventing catastrophic losses due to shortened replication on lagging strand.
Telomerase ads approximately 1000 copies of DNA sequence at each chromosome end. Acts on germ cells and only some somatic cells (bone marrow and intestinal epithelium)
Programmed cell death occurs when telomere is determined to be too short.