Study questions.

1. Which of the following nucleotide substitutions are transition mutations?

a) G for A

b) A for C (transversion)

c) C for T

d) T for G (transversion)


2. Which of the substitutions in question 1 are transversion mutations?



3. Explain why most nucleotides that have been misincorporated during DNA synthesis in E. coli do not lead to mutant progeny. Because the editing 3'-exonuclease of DNA polymerase III and the Methyl-directed Mismatch Repair pathway usually remove the errors allowing for replacements with correctly incorporated nucleotides.

4. Match the type of mutation or physiologic consequence in the right column with the appropriate mutagen in the left column. You may have to look up some of these compounds.

a) 5-Bromouracil 2

1) Transversion.


b) 2-Aminopurine 2

2) Transition.


c) Hydroxylamine 2

3) Insertion or deletion.


d) Acridines 4,5_____________

4) Translational frameshift.


e) Nitrous acid ________2 _______

5) Block in replication.


f) Benza[a]pyrine________4,5_ ____

EXPLANATION: Base analogues like 5-bromouracil and 2-aminopurine incorporate in place of the corresponding natural bases and usually lead to transition mutations when they exist in ionized or native states that have different base-pairing properties from the natural bases. The compound 5-bromouracil incorporates quite well in place of thymine (5-methyluracil) and codes like thymine (i.e., pairs with adenine in subsequent replications); however, it occurs in the ionized form more frequently than does thymine and as a consequence, creates more A:C and C:A misincorporations during replication. On subsequent replications these misincorporations become transition mutations. The compound 2-aminopurine incorporates, at low levels, in place of either guanine (2-amino, 6-oxypurine) and is a highly potent mutagen that causes G:T and T:G misicorporations, i.e., it is also a transition mutagen.

Compounds that lead to oxidative deaminations (by reacting with the NH2 groups of A, G, and C) lead to transitions. They in essence change the H-donating side group (NH2) to an H-accepting (=O) side group. Hydroxylamine and nitrous acid belong to this category of mutagens.

Planar, hydrophobic compounds like acridine dyes and Benza[a]pyrine intercallate in DNA and increase the frequency of slippage during DNA replication, thus leading to frameshift mutations. The presence of an intercalating agent in double-stranded DNA may cause a pause in replication that is usually overcome through the slippage that leads to frameshifts.


5. If DNA polymerase makes a mistake, thus creating a pair of incorrectly hydrogen-bonded bases, the mistake is corrected by a special _mismatch repair_______ system that uses methylation to distinguish new strands from old.

6. The enzyme that replicates the HIV virus is ____reverse transcriptase, which lacks 3'-exonuclease (editing)_activity and consequently has poor fidelity.

7. When acting on replicated DNA, the methyl-directed mismatch repair system in E. coli can distinguish the parental strand from the progeny strand as long as one or both are methylated, but not if both strands are unmethylated. True or false?

ANSWER: False. This repair system works only if one strand is methylated at GATC sequences and the other not.

8. Which of the following statements about DNA polymerase III holoenzyme from E. coli are correct?

a) It elongates a growing DNA chain approximately 100 times faster than does DNA polymerase I. Pol III is more processive than pol I.

b) It associates with the parental template, adds a few nucleotides to the growing chain, and then dissociates before initiating another synthesis cycle. This is what happens with a nonprocessive enzyme.

c) It maintains a high fidelity of replication, in part by acting in conjunction with a subunit containing a 3' Õ 5' exonuclease activity.

d) When replicating DNA, it is a molecular assembly composed of at least 10 different kinds of subunits. The b subunits (b dimer) of this assembly is the "processivity" factor.

ANSWER: A, C and D