lecture 8: nuclease enzymes

hydrolysis of the phosphodiester bond in an SN2 reaction:

• Substitution: AB + C <=> AC + B

• nucleophile: donates an electron lone pair

• 2 molecules: bimolecular

the general schemem for hydrolysis of nuceic acids mechanism

mechanism for which RNA phophodiester bond self-cleaves under alkaline conditions

describe the general mode of action for restriction endonucelases

1. the enzyme non-specifically binds randomly onto the DNA strand

2. the active site aligns with the phosphodiester bond it is designed to cut

3. nucleophilic attack with water to break the bond

4. after both strands are cut, the enzyme is released

what are the 2 most well characterised type II restriction endonucleases (REases)

EcoRI and EcoRV

explain the signature motif in EcoRI and EcoRV

PD…D/ExK

• the proline and aspartate are involved in positioning the backbone

• the aspartate/glutamate and lysine coordinates the magnesium ion

what are the reasons that REases need metal ions to enable the catalytic process?

• enhance the deprotonation of the attacking water (nucleophile)

• stabilises the negative charge on the pentavalent intermediate state of the phosphate

• could provide some stabilisation to the leaving 3’ oxyanion

what metal ions can REases use?

• Mg2+

• Mn2+

• Ca2+ (but could inhibit the enzyme)

one metal ion mechanism

2 metal ion mechanism

HNH-family nucleases

contains a number of conserved histidine and asparagine residues

they use metal ions to faciliate the phosphodiester bond cleavage (can use Mg²⁺, Ni²⁺, Co²⁺, Zn²⁺)