lecture 5 - denaturation & mutagenesis

16 slides

what are 4 steps of DNA denaturation?

1. covalent bonds remain intact

2. H-bonds are broken; 2 strands separate

3. base stacking is lost

4. we see hyperchromic effect/shift; UV absorbance increases

what can denaturation be induced by?

high temp or change in pH

what are 4 factors of thermal DNA denaturation?

1. DNA exists as a double helix at normal temp

2. DNA strands dissociate at elecated temps and re-annealed when temp is lowered

3. this reversible thermal denaturation is basis of PCR

4. DNA denaturation is commonly monitored by UV spectrophotometry at 260 nm

what are 5 factors affecting denaturation?

1. base composition

2. presence of protein

3. size of DNA sample

4. pH (adjust presence/absense of proton)

5. ionic strength

what are 3 factors that Tm depend on?

1. base composition → increase GC = increase Tm

2. DNA length → longer DNA = increase Tm

3. pH/ionic strength → high salt = increase Tm

what temp do AT rich regions melt at?

lower temperature than GC rich regions

what are 2 factors of deamination?

1. deamination reactions are slow but occur for large number of residues

2. so net effect is significant

what are 3 points of depurination?

1. depurination is hydrolysis of N-glycosidic linkage

2. significant for purines: about 10,000 purines lost per day in mammalian cell

3. results in apurinic site

what is oxidative damage?

hydroxylation of guanine

• mitochondrial DNA is most susceptible to this → more oxygen; DNA is not as tightly packed

what is chemical alkylation?

methylation of guanine

what are 2 points of radiation induced mutagenesis?

1. UV light induces dimerization of pyrimidines

2. ionizing radiation (X & Y rays) cause ring opening and strand breaking

*cells can repair some modification, but an accumulation is linked to aging and carcinogenesis

what are 3 other functions of nucleotides?

1. energy source

2. coenzymes

3. regulatory molecules