DNA Damage and Repair

DNA blueprint

DNA susceptible to change

Plus factor driving force in evolution Minus factor too much change is lethal Applies to all living organisms

Why study genetic variation in bacteria?

Effect of Chromosomal structure on damage

Prokaryotes Unicellular Single circular Naked

Eukaryotes Multicellular Multiple DNA copies Histones Core regions and linker regions

Genetic variation in bacteria

Rapid growth

increase from 1 109 in 12 h

Approx 1 in every 109 cells is mutant Can see 1 mutation event in 12 h Changes easily seen

Consequences of mutations
1. General

Bad: Antibiotic resistance Good: survival in ecological niche industrial processes (biotechnology)

2. Scientific value

Useful tools (eg lactose operon)

gene regulation assign function to genes

Definitions

Genotype:

Genetic information on the genome

Phenotype:

Physical characteristics

Wildtype:

Not altered with respect to the particular genotype or phenotype being examined.

How does damage occur?

Induced errors

External factors Contact with damaging agent in the environment - Chemical - Physical Natural errors - normal cell processes Metabolism By-products of metabolism (eg: free radicals) Replication Incorrect proofreading by DNA polymerase

Spontaneous

eg DNA replication = 1 in 109

Types of mutations

Large disruptions to genome

Transposons Duplications Large deletions Chromosomal inversions

Smaller changes (1 or 2 bases)

Causes of mutations

UCT

How are mutations caused?

Induced Chemical Chemicals Pollutants

Physical Radiation (UV, Gamma)

DNA structure and mutations

Important features of DNA

1) Specific base pairing 2) Strand separation during replication 3) Integrity of DNA helix sugar phosphate backbone during strand separation

DNA DAMAGE
SITE BASE PAIRING TYPE ALKYLATION BASE ANALOG RADIOLYSIS INTRA-STRAND CROSS-LINKS STRAND SEPARATION INTER-STRAND CROSS-LINKS E.G. MNNG 5-FU X-RAYS MTC UV CIS-Pt MTC CIS-Pt

BACKBONE

STRAND BREAK X-RAYS

Types of chemical agents

i) Base analogues Cause inaccurate base pairing Sometimes used in cancer therapy Eg: a) Adenine analogue 2 amino purine b) Thymine analogue 5 fluorouracil

Types of chemical agents

ii) Base modifiers

Nitrous acid (HNO2) - causes deamination of adenine

Adenine convert to hypoxanthine

Can pair with C

reacts with C (hydroxylation), pairs with A Transfer alkyl groups to bases (methyl, ethyl etc) AlkG pairs with T Eg: Nitrosoguanidine

Hydroxylamine (NH2OH)

Alkylating agents

Ethyl methane sulphonate

Nitrogen mustard (gas chemical warfare)

Types of chemical agents

iii) Intercalating agents
Cause frameshift mutations

ethidium bromide

3 ringed molecule Intercalates (inserts) into DNA helix Changes distances between bases Additional bases incorporated Back-mutation

Types of chemical agents

iv) DNA crosslinkers

Insert between strands of helix Form covalent bonds

Strands cannot separate during replication

Eg:

Mitomycin C bifunctional alkylating agent

Cis platinum
Psoralen