Mutagens and Their Modes of Action

Chapter 13, P.O.G.

Types of changes that mutagens can induce

a) Transitions - one purine is replaced by another or one pyrimidine is replaced by another

e.g.   A
mutation A*

T
T

- where A* has the base pairing properties of guanine.

At the first round of replication A* will pair with CYTOSINE instead of thymine. In the next round of replication, cytosine pairs with GUANINE so G replaces adenine

.........A*

.........C

..........A*

..........C

.........C

.........G

This is an A to G (A ---> G) transition.

Also: G ---> A; T ---> C and C ---> T transitions.

 

b) Transversions - a purine is replaced by a pyrimidine or vice versa.

e.g. A ---> C; G ---> C

- transversions lead to purine-purine mispairs and pyrimidine-pyrimidine mispairs within the DNA. This can significantly distort the sugar-phosphate backbone of the DNA (purines are larger molecules than pyrimidines).

There are 8 transversions and 4 transitions.
 
 

Modes of Action of Standard Mutagens

a)   Nitrous Acid (HNO₂). This deaminates nucleotides removing amino (-NH₂) groups and substitutes keto (=O) groups.

e.g. Cytosine is deaminated to uracil which pairs with adenine which then pairs with thymine. The result is a C--- T transition.
Also, adenine is deaminated to HYPOXANTHINE which resembles guanine and pairs with cytosine which pairs with guanine giving an A --- G transition.

HNO₂ then gives AT --- GC transitions.

b)   Hydroxylamine (H₂NOH or NH₂OH).. Reacts with pyrimidines in DNA, probably mutagenic only with cytosine. Results in N⁶-hydroxy cytosine which pairs with adenine producing a C --- T transition. GC — AT transitions only.

c)   Alkylating agents. Diverse, highly reactive (nasty) chemicals that introduce alkyl groups (CH₃-, CH₃CH₂- etc) into nucleotides. Very potent.

- main targets for alkylation in DNA are N and O atoms of the bases, e.g. N⁷ of guanine; O⁶ of guanine.

- O⁶-alkylguanine and O⁴alkylthymine most likely to undergo mispairing.

O⁶-alkylguanine pairs with thymine giving G — A transitions

O⁴alkylthymine pairs with guanine to give T — C transitions

Alkylating agents may be monofunctional (have only one alkyl group to donate) or bifunctional.
 

Alkylating agents include:

NITROGEN MUSTARD -  di-(2-chlorethyl)methylamine

MUSTARD GAS (sulphur mustard) -  di-(2-chlorethyl)sulphide

DIETHYL SULPHATE (C₂H₅)₂SO₄

METHYLMETHANE SULPHONATE (MMS)  CH₃-O-SO₂-CH₃

ETHYLMETHANE SULPHONATE (EMS)  C₂H₅ CH₃-O-SO₂-CH₃


N-METHYL-N'-NITRO-N-NITROSOGUANIDINE (MNNG, NTG)

bifunctional alkylating agents, e.g. mustard gas, may also induce crosslinking of DNA strands leading to chromosome breaks & loss of chromosome segments.

d)   Heat. Can deaminate cytosine to uracil giving C —> T transitions. Also produces
      G —> C transversions by an unknown mechanism.
      the majority of heat-induced mutations are subsequently repaired.

e)   Base analogues. Have similar chemical structures to naturally occurring bases but with critical modifications. Exert mutagenic effect by being incorporated into DNA during replication in place of the bases whose structure they mimic.
The two most widely used:
5-BROMOURACIL (5-BU, 5-BrDU) a thymine analogue, ( — 5-bromodeoxyuridine nucleotide)

2-AMINOPURINE, (2-AP) adenine analogue
base analogues are probably mutagenic because of ambiguity of their base pairing due to tautomeric shifts.
e.g. 5-BU in its rare enol form is incorporated oposite guanine during replication, then functions in subsequent replication as thymine (in normal keto form), causing
GC  —>  AT transitions.
reverse is also possible, so GC —> AT transitions.
2-AP can pair with cytosine as well as thymine (by tautomeric shifting) leading to same transitions.

methylated cytosine bases in DNA form stable bonds with 2-AP, also leading to mutation.

f)   Acridine compounds e.g. PROFLAVINE
- induce frameshifts

- the ring system mimics a H-bonded DNA base pair in shape and hydrophobicity

- intercalates (inserts) between stack of base pairs in DNA helix

- many acridines are more mutagenic than proflavine and combinations like the ‘ACRIDINE-HALF MUSTARDS’ (acridines coupled to alkylating agents) have been developed.

g)   Ionising radiation (X-rays, (gamma-rays, "alpha-particles, & neutrons)
- these generate charged atomic and molecular particles by displacement of electrons

- induce chromosome breaks and rearrangement. X-rays lead to point mutations.

- most studies of effect of ionisation radiation were made with X-rays. In the low dosage range he mutagenic effects are directly proportional to the dosage
Fig - Relationship between irradiation dosage and mutation frequency in Drosophila
- % sex-linked lethal mutations in Drosophila is easy to assay.

- the Roentgen unit (r) is based on the number of ionisations produced in 1 cm³ of air under standard conditions.
From the figure:
- exposure to low doses for long times is as mutagenic as a single exposure at moderate dosage, i.e. X-ray dosage is cumulative in the low dosage range

- this implies there is no safe lower limit or threshold below which ionising radiation has no mutagenic effect.

X-ray induced damage is thought to be brought about by free radicals (Molecules that contain an atom with an unpaired electron) that are highly reactive

- free radicals of oxygen are thought to be induced intracellularly by X-rays

- when Drosophila pupae irradiated with X-rays in an atmosphere of O₂ or N₂, the yield of chromosome and X-linked recessive lethals is 3 times higher in O₂ (aerobic) than in N₂ (anaerobic).

h)   Ultraviolet light
- primary effect is crosslinking between pairs of adjacent pyrimidine residues to give PYRIMIDINE DIMERS (cyclobutane pyrimidine dimers)

- thymine dimers are the most stable and plentiful type from UV irrad, but C dimers and mixed C-T dimers also occur with frequencies depending on DNA base composition

- in many organisms studied including E. coli but not humans, pyrimidine dimers are efficiently dealt with by PHOTOREACTIVATION.

- this depends on the enzyme photolyase (encoded by phr in E. coli) which reverses dimerisation to restore normal DNA structure.

- the anzyme uses visible light as a source of energy. Photolyase can associate with the dimers in the dark, but must absorb a photon of blue light before it can function.

Photoreactivation is a DNA repair mechanism - more later
 

Some mutations are reversible by mutagens
e.g. nitrous acid causes AT —> GC transitions. 5-BU can cause AT —> GC only whereas hydroxylamine causes can cause CG —> AT only

- so if a mutation can be reversed by hydroxylamine but not by 5-BU, then the original mutation was AT —> GC

- if 5-BU but not H₂NOH can reverse a mutation, then the mutation was GC —> AT.

The effects discussed are direct mutagenic effects, but mutagens have other effects:

a)   Non-hereditarye.g. the majorreaction product of many alkylating agents is N⁷-alkylguanine, but this behaves like guanine and is not mutagenic

b)   Inactivating - prevents transmission of the altered genome from parent to offspring.
- e.g. HNO₂ and some alkylating agents crosslink DNA strands so they cannot be separated for replication etc. (- may be repaired by recombination repair).
c)   Premutational lesions
- inactivating type lesions may be repaired. In some cases repair is incorrect (misrepair) and incorrect nucleotides are incorporated

- so mutagen-induced damage is not the absolute cause of mutation - the cause is error in the repaair process

- hence the damage caused by the mutagen is considered the premutational lesion

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