2. Nucleic Acids and Gene Expression

Question 1

Describe the process of DNA replication.

Answer 1

• free dNTPs synthesised in cytosol before being transported to nucleoplasm through nuclear pores in nuclear envelope
• replication begins at oriR with specific sequence of nucleotides; initiator proteins bind and recruit helicase that unwinds and unzips DNA by disrupting H bonds between complementary base pairs, using ATP
• single strand binding proteins (SSBPs) bind to single-stranded regions, preventing strands from reannealing
• topoisomerase relieves strain ahead of replication fork by creating transient break in sugar-phosphate backbone by disrupting phosphodiester bonds; enables free rotation and unwinding for initiation
• replication forks spread bidirectionally to form replication bubble; serve as templates

Question 2

Synthesis of new DNA strands

Answer 2

• DNA primase adds RNA primer to each parental DNA strand; provides free -OH so DNA polymerase can initiate DNA synthesis; 5’→3’ direction
• DNA polymerase selects dNTPs with nitrogenous bases complementary to those on the parental strands
• each dNTP loses pyrophosphate and hydrolysis of pyrophosphate bond is coupled with phosphodiester bond formation
• as DNA moves along template, part of it proofreads previous region to ensure proper base pairing; removes incorrect deoxyribonucleotide through 3’→5’ exonuclease activity and replaces it
• different DNA polymerase removes RNA primer and replaces it with DNA; DNA ligase seals nick through phosphodiester bond formation

Question 3

Describe how the end-replication problem arises.

Answer 3

• DNA replication begins at oriR and occurs bidirectionally
• after last RNA primer is removed by ANOTHER DNA polymerase, as there is no DNA polymerase that can recognise the free 5’ phosphate end and add a deoxyribonucleotide without availibility of a free 3’ -OH, each round of DNA replication forms increasingly shorter DNA molecules
• 3’ overhang formed