Lec 3 - Hydrolysis of phosphodiester bonds

Question 1

Draw out the RNA backbone including which phosphodiester bonds are going to be cleaved also give the names of the carbon groups where cleavage occurs

Answer 1

Raff 3 pg 2 3’ and 5’

Question 2

What type of reaction is the hydrolysis of the phosphodiester bonds

Answer 2

SN2 substitution nucleophillic bimolecular

Question 3

Draw out the general scheme for the hydrolysis of nucleic acids for an SN2 nucleophillic attack

Answer 3

Question 4

Draw out the associative and dissociative SN2 mechanisms involving the P group

Answer 4

Raff 3 pg 5

Question 5

Under what conditions can the phosphodiester bond self cleave? Draw out this mechanism

Answer 5

alkaline

Question 6

Are nucleases separate enzymes or part of larger complexes?

Answer 6

can be both separate and part of larger complexes - eg DNAP has a nuclease domain as a checking mechanism

Question 7

Describe the general mode of action of REases

Answer 7

• nonspecific binding to DNA - linear diffusion - by either a scanning mechanism or jumping - increase in no. specific interactions @ target site creates fold in DNA - metal ions act as cofactors - hydrolysis - release of products

Question 8

Give 2 examples of Type II REases and give the residue sequence of the common motif found in their AS

Answer 8

EcoRV EcoRI - PD…D/ExK

Question 9

Give examples of metal ion cofactors found in REases

Answer 9

• Mg2+, Mn2+ (less common because rare) - Ca2+ most likely to be used as an inhibitory compound

Question 10

Give the 3 uses that metal ion cofactors have

Answer 10

• stabilises the double -ve charges of the pentavalent TS - promotes formation of OH- nucleophile by a base eg Lys/Glu - stabilises the 3’ Oxyanion leaving group

Question 11

How are the metal ions stabilised in the AS

Answer 11

• Asp/Glu residues

Question 12

Why is Mg2+ a popular metal ion used in REases?

Answer 12

high charge density and can coordinate H20 molecules

Question 13

Where is at least one metal ion positioned w/in the AS

Answer 13

near the scissile P group which fills the space when the p group leaves

Question 14

Describe the one metal ion mechanism

Answer 14

substrate assisted cleavage

• O- group from nearby P is used to generate attacking OH- nucleophile

Question 15

Which compound and why was it used to identify this one metalion mechanism

Answer 15

methylphosphonate no -ve charge on O- therefore no nucleophile could be generated

Question 16

Describe the 3 functions of the Mg2+ ions in a 2 metal ion catalysis

Answer 16

• together stabilise pentavalent TS negative charges (need to be 4A apart for this) - one promotes the formation of the nucleophile - one positions a H20 molecule to attack as H+ donor to stabilise the 3’ oxyanion group

Question 17

Describe the HNH family nucleases

Answer 17

uses His residues to promote the formation of the OH- nucleophile uses Mg2+ to stabilise the TS intermediate

Question 18

Give the name of a HNH type nuclease and name a couple of metal ions that can be used as cofactors

Answer 18

E7 colison His molecules (as well as generating nucleophile) stabilise the positioning of Zn2+, Mg2+

Question 19

Describe the 2 pathways that can be followed to remove the ssDNA flaps from the Okazaki fragments on the lagging strand

Answer 19

RNase H dependent - RNase H then cleavage by FEN1 (humans) RNase H independent - just cleavage by FEN1

Question 20

Describe how the T5 phage FEN breaks the flap ssDNA

Answer 20

helical arch structure where the flap ssDNA is passed through allows the 3 metal ions in its AS to be in close proximity to the P backbone the metal ions (Mg2+) stabilise the TS intermediate and promote the formation of the attacking OH- nucleophile