Nucleases

Question 1

What do nucleases do?

Answer 1

cleave (hydrolyse) phosphodiester bond via SN2 rxn

Question 2

What are the two models of the mechanism of nucleases?

Answer 2

Associative
-attacking water looses H+ to form attacking nuc
-pentavalent phosphate intermediate
Disassociative
-leaving group leaves before nuc attacks
-trigonal arrangement intermediate

Question 3

How do DNA restriciton endonucleases work?

Answer 3

-bind non-specifically to DNA
-scans along DNA to specific site
-coupling
-catalysis (using metal ion)
-releases prod

Question 4

What do metal ions do in catalysis?

Answer 4

-enhance protonation of nucleophile (attacking water in nucleases)
-stabilise -ve charges on intermediates (pentavalent phosphate in nucleases)
-stabilising leaving group (3’ oxyanion in nucleases)

Question 5

Mechanism of one metal ion catalysis

in nucleases

Answer 5

substrate-assisted cleavage
-metal ion stabilises leaving group

Question 6

Mechanism of two metal ion catalysis

in nucleases

Answer 6

Ion 1 - facilitates deprotonation of nucleophile and positions it
Both ions - stabilise -ve charge of intermediate
Ion 2 - interacts with leaving group

Question 7

Why can RNA self-cleave but DNA can’t?

Answer 7

-RNA has extra OH
-in alkaline conditions, OH acts as nucleophile
-base removes H from OH, so that O can attack nearby OH in phosphodiester bond

-DNA doesn’t have that OH (only H) so can’t -always requires endonucleases (N/B: RNA also has nucleases eg. RNaseA)