Enzymology of DNA replication

Question 1

write a note on the first DNA polymerase enzyme

Answer 1

1. synthesises DNA through polymerisation at the free 3’OH
2. has a 3’ to 5’ exonuclease activity which is important for proofreading newly synthesised DNA - can fix mistakes
3. has 5’ to 3’ endonuclease activity that can degrade a strand ahead of the advancing polymerase such as the laid down RNA primers and in DNA repair

Question 2

what are klenow fragments

Answer 2

a large domain on the polymerase enzyme on which DNA synthesis and exonuclease activities are carried out

Question 3

write a note on DNA polymerase III of E coli

Answer 3

has 3 subunits that comprise the core of the enzyme:
alpha - has the DNA polymerase activity
epsilon - has the 3’ to 5’ exonuclease that removes mismatched bases when theyre incorporated (1 error every 1000 nts)
theta subunit stimulates the epsilon subunit proofreading

Question 4

write a note on eukaryotic DNA polymerase

Answer 4

not much is known. multiple DNA polymerases participate (around 15)
alpha - priming the synthesis
delta - elongating the lagging strands
epsilon - leading strand replication
beta - repair
gamma - maybe be involved in replicating mitochondrial DNA

Question 5

what are some other enzymes and proteins used in DNA replication

Answer 5

helicases - use ATP energy to separate parental strands

primase - binds to helicase to form a primosome. primase is activated by helicase and makes a short RNA primer that new nucleotides can be added onto

topoisomerase 1 and DNA gyrase (a type 2 topoisomerase) winds and unwinds supercoiled DNA to facilitate DNA replication

single strand DNA binding protein
binds to separated DNA strands and coats them so they cannot re-anneal