DNA 2 - enzymes

Question 1

Central Dogma for DNA

Answer 1

DNA makes RNA makes Protein

Question 2

Endonucleas and exonuclease similarity and differences

Answer 2

Both cleave phosphodiester bonds -> endo cleaves internally, exo at the end of a polynucleotide chain

Question 3

Significant of restriction enzymes - what do they recognise?

Answer 3

Very specific endonuclease which cuts at a specific sequence. Therefore recognises inverted repeats on anti parallel DNA strand

Question 4

DNA replication is

Answer 4

semi-conservative

Question 5

PCR overview

• reagents
• order of steps

Answer 5

Thermo-stable taq polymerase used +
Customised primers + free nucleotides
-Denaturation 96°c
-Primer annealing 55°c
-Primer extension 72°c
each cycle doubles DNA content

Question 6

DNA Polymerase I functions and characteristics

Answer 6

Polymerisation activity 5’-3’
Exonuclease activity both 3’-5’ and 5’-3’
requires primer with free 3’-OH and template strand
-replaces RNA primers with DNA
-carries out DNA repair/error correction

Question 7

DNA Polymerase III activity, functions and characteristics

Answer 7

Polymerisation activity 5’-3’
exonuclease activity only 3’-5’
requires primer with free 3’-OH and template strand
-High rate of processivity facilitates synthesis of large stretches of DNA

Question 8

How is process of replication initiated?

Answer 8

DNA helicase separates DNA strands, forming replication forks at ‘origin’ which both converge at termination site.

Question 9

DNA pol III activity following formation of replication fork?

Answer 9

pol III forms long strand on leading strand 5’-3’. RNA primase adds short RNA primers to lagging template strand. DNA pol III polymerises 5’-3’ between primers forming ‘okazaki fragments’. DNA pol III is associated via clamp-loading protein.

Question 10

DNA pol I activity following this and other present enzymes?

Answer 10

DNA pol I replaces RNA primers with DNA, process results in ‘nick translation’. DNA ligase seals up nicks in the phosphate backbone.