Genetics Chapter 13

Which of the following amino acid substitutions should you expect to be induced by 5-bromouracil with the highest frequency? (a) Met —> Leu; (b) Met —> Thr; (c) Lys —> Thr; (d) Lys —> Gln; (e) Pro —> Arg; or (f) Pro —> Gln? Why?

(b) Met —> Thr. 5-Bromouracil induces transitions, not transversions. All other changes listed require transversions

Hydroxylation

-NH2OH -causes cytosine to pair with adenine -unidirectional transition GC--> AT

Light-dependent repair

-absent in mammals -DNA photolyase recognizes dimers and uses light to cleave

Mismatch

-backup for proofreading -knows which base is correct because newly replicated DNA is not methylated -A in GATC sequence is normally methylated -MutH (has endonuclease), MutL, MutS -requires exonuclease -Pol III and ligase fill

base analog

-have structures similar to normal bases -increase mispairings through tautomeric shifts -cause transitions -only occur during replication

Deamination

-nitrous acid -deaminate A to bind to C, C to bind to A, and G to bind to C -bidirectional transitions AT<-->GC

Acridines

-proflavin, acridine orange, ethidium bromide -intercalate between bases -cause frameshift mutations during replication

Five mechanisms of E.coli DNA repair 1. 2. 3. 4. 5.

1. Light-dependent repair 2. excision repair (BER and NER) 3. mismatch repair 4. postreplication repair 5. error-prone repair (SOS)

tautomeric shifts cause which kind of bonds

A:C and G:T

The bacteriophage T4 genome contains about 50 percent A:T base pairs and 50 percent G:C base pairs. The base analog 2-aminopurine induces A:T —> G:C and G:C —> A:T base-pair substitutions by undergoing tautomeric shifts. Hydroxylamine is a mutagenic chemical that reacts specifically with cytosine and induces only G:C —> A:T substitutions. If a large number of independent mutations were produced in bacteriophage T4 by treatment with 2-aminopurine, what percentage of these mutations should you expect to be induced to mutate back to the wild-type genotype by treatment with hydroxylamine?

About half of the induced mutations would be expected to mutate back to the wild-type genotype

5-bromouracil

base anolog causes bidirectional transitions AT<-->GC

SOS (error prone repair)

desperate attempt to escape lethal situation, gaps eliminated but errors occur

Ames Test

developed by Ames to determine if substances are mutagenic/carcinogenic by seeing if they revert mutanations to His gene

usefulness of mutations

essential to provide new genetic variability

Why was the Ame test altered?

some chemicals that mutate bacteria are altered by liver to be harmless and some chemicals are not carcinogenic until the liver alters them

NER

-2 UvrA proteins and 1 UvrB recognize T dimer, binds opposite -Uvr A leaves, Uvr C binds and makes 5' incision -UvrB makes 3' incision, UvrD (helicase II) removes -DNA Pol and Ligase seal

BER

-DNA glycosylases (specific) recognize abnormal bases -glycosidic bond cleaved making AP site -AP endonuclease and phosphodiesterases remove remaint phosphate-sugar groups -DNA polymerase and ligase repair gap

How do acridine-induced changes in DNA result in inactive proteins?

Mutations induced by acridine dyes are primarily insertions or deletions of single base-pairs. Such mutations alter the reading frame (the in-phase triplets specifying mRNA codons) for that portion of the gene distal (relative to the direction of transcription and translation) to the mutation. This would be expected to totally change the amino acid sequences of polypeptides distal to the mutation site and produce inactive polypeptides. In addition, such frameshift mutations frequently produce in-frame termination codons that result in truncated proteins.

How does the action and mutagenic effect of 5-bromouracil differ from that of nitrous acid?

Nitrous acid acts as a mutagen on either replicating or nonreplicating DNA and produces transitions from A to G or C to T, whereas 5-bromouracil does not affect nonreplicating DNA but acts during the replication process causing GC <—> AT transitions. 5-Bromouracil must be incorporated into DNA during the replication process in order to induce mispairing of bases and thus mutations

How does nitrous acid induce mutations? What specific end results might be expected in DNA and mRNA from the treatment of viruses with nitrous acid?

Nitrous acid brings about a substitution of an OH group for an NH2 group in those bases (A, C, and G) having NH2 side groups. In so doing, adenine is converted to hypoxanthine, which base-pairs with cytosine, and cytosine is converted to uracil, which base-pairs with adenine. The net effects are GC <—> AT base-pair substitutions

Is a mutation random or directed by environmental stress?

No

Are mutational changes induced by nitrous acid more likely to be transitions or transversions?

Transitions

Would you expect nitrous acid to induce a higher frequency of Tyr —> Ser or Tyr —> Cys substitutions? Why?

Tyr -> Cys substitutions; Tyr to Cys requires a transition, which is induced by nitrous acid. Tyr to Ser would require a transversion, and nitrous acid is not expected to induce transversions.

tautomeric shifts

keto and amino forms have a less common enol and imino forms which cause mispairing

germinal mutation

occur in germ-line cells, affecting progeny

somatic mutation

occurring in somatic cells

spontaneous mutation

occurring without known causes

Robin Holiday

proposed recombination mechanism where endonuclease cleaves single strand and this strand displaces single strand on different DNA

Transitions

purine > purine or pyrimidine > pyrimidine, caused by tautomeric shifts

Transversion

purine > pyrimidine

Gene conversion

recombination coupled with mismatch repair using one gene for correct template to fix a second template

induced mutation

resulting from exposure to physical or chemical agents

Postreplication Repair

utilizes recombinaton to provide sequence needed to fill gaps that are not repairable; t-dimers or obstructions not repaired