http://www.unc.edu/courses/2009spring/envr/740/001 slide 34
Begin 02/17/09
REPAIR
Of obvious interest in a discussion of chemical carcinogenesis is the manner in which DNA damage is dealt with by cells. Any damage to DNA is called a lesion, whether it results from reaction with exogenous chemicals or from endogenous errors, which result in mispairing of bases. Mismatches can be caused by faulty proofreading, but also by structural isomerization by the bases themselves- a phenomenon called tautomerization. Tautomerization is illustrated by the example of Gua, which is the base most susceptible to mismatching by tautomerization. While
O of the proton from N1 to O
6 which the 6-membered ring is aromatized O
[OH; keto-enol]
H
2
N
HN
N
N
N
H
N
O
H
2
N
H
2
N
N
N
N
H
2
N
N
OH
N
N
N
O OH
O
. This shift changes the pairing properties of Gua, so that it behaves like Ade:
N N
N
N N
H
H
H
2
N N
N
H
2
N N
N
O
H
N
HN
O
N
N
N
H
3
C
O
N N
H
H
O
O
N
N
H
HN N
N and pairs with T rather than C. If this mismatch is not recognized and repaired, one round of replication on the strand bearing the erroneously incorporated T will result in a mutation, as
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the next overhead shows. This process, in which the mutation has been made permanent, is called “fixing” the mutation. I should point out that tautomerization is a chemical change so this mutation is chemical.
[OH; point mutations] G

T

G

C + A

T
The type of mutation just described is called a point mutation because a single base pair on the
DNA is changed. When a purine is exchanged for purine, or a pyrimidine for pyrimidine, the point mutation is called a transition mutation. If purine is exchanged for pyrimidine:
G

C

C

G, T

A

A

T, G

C

T

A the point mutation is called a transversion mutation. Point mutations may change a single codon
(unless they occur in position 3 of a redundancy). When one of the termination codons is generated by the mutation, the mutation is called a nonsense mutation, and when the amino acid is changed, it is a missense mutation. If a missense mutation occurs at a critical residue- for example a turn in the protein backbone is eliminated by substitution for a Pro, or a disulfide linkage is removed by substitution for a Cys, and the protein fails to fold properly, then function may be completely eliminated or changed. On the other hand, the substitution could occur in a completely innocuous manner and not change function at all, or the effect could be intermediate.
Similarly, a nonsense mutation, if it is efficiently recognized during translation- efficiency of termination is a function of context and not all termination codons are efficiently recognized - a truncated protein will result. If the truncation occurs at a critical location; for example, at the
DNA binding domain of the lac repressor or at the C-terminal, which is responsible for the oligomerization, then the protein will be non-functional. On the other hand, the termination may occur in a region that does not affect function.
There is a second type of mutation that results when one or more bases are deleted or inserted into the genome. For any change not divisible by 3, the entire reading frame beyond the point of insertion or deletion will be shifted - hence this type of mutation is called a frameshift . If n bases
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are inserted, then the lesion is a +n frameshift; if n bases are deleted, it is a -n frameshift. In practice, the term frameshift is applied only when n= 1 or 2. Illustrated in the next slide is an insertion of a G (red) in the codon GCC.
[Overhead: illustrate frame-shift mutation]
Frameshifts are common in runs of 4 or more G

C pairs, and the mechanism of the error appears to involve the looping-out of the daughter strand to give insertions or the template strand to give deletions as the bottom panel illustrates.
[OH; proposed mechanism of frame-shift]
3

-G-p-G-p-G-p-G-p-G-p-G-5

5

-C-p-C p-C-OH
| |
p-C
Insertion
3
5


G-p
| |
-G-p-G-p G-p-G-p-G-p-G-5
-C-p-C-p-C-OH
 frameshift]
Deletion
Since runs of GC pairs are common and are sites of frame shift mutations, such sites are called mutational “hotspots”.
Mutations in mRNA resulting from mutations of DNA are relatively frequent, so cells have evolved post-transcriptional mechanisms to correct them. The simplest systems are nonenzymatic, and were developed to cope with both nonsense and missense point mutations. The strategy, especially in prokaryotes, involves generation of tRNAs bearing amino acids in which the anticodon has been mutated to recognize the mutant codon. Such mutant tRNAs are called suppressor tRNAs. The best characterized suppressors are bacterial suppressors of nonsense mutations. Bacterial nonsense suppressors characterized from E. coli are shown on the next overhead.
[OH; table of nonsense suppressors]
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The termination codons are called amber, ochre or opal because they were originally detected as mutations in the drosophila gene coding for eye color of drosophila that produced the corresponding colors. UAG, called the amber codon, is recognized by 6 tRNAs having the anticodon CUA← and carrying the amino acids shown in the table, depending on the mutation carried in the anticodon. Five suppressors with the anticodon UUA← suppress the ochre mutation (UAA), but also recognize the amber codon (UAG) as predicted by the Wobble hypothesis (U in position 1 of anticodon pairs with A,G in position 3 of codon). Two suppressors recognize the opal termination codon UGA. One suppressor carrying Trp continues to recognize the normal codon UGG for Trp presumably consistent with the expected Wobble pairing. Eukaryotes also use suppressor tRNAs to suppress nonsense mutations, although much less is known about eukaryotic systems. The mechanisms of suppression are not as straight forward as for prokaryotes, and involve inclusion or modified bases such as pseudouridine, 3methyl-C, queuosine (a modified G) in the anticodon loop as well as elsewhere in the tRNA, all of these substitutions act to alter the flexibility of the tRNA to enable recognition of the various stop codons. Context is also a very important factor in efficiency of stop codon recognition, especially on the 3′ side of the nonsense codon.
Missense mutations are compensated for by the same strategy, except that the mutant tRNA anticodon recognizes the missense codon, rather than a termination codon. A missense suppressor has been identified that will suppress the mutation of wild-type codon GGA (coding for Gly) to AGA which would normally be translated as Arg. The suppressor carries Gly with the anticodon UCU←. Suppression is only partial, because both the suppressor and the normal
Arg tRNA respond to AGA. Furthermore, UCU
Gly←
recognizes the codon AGG for Arg. In yeast, substitution in the UGC anticodon of tRNA-Ala is altered to UGU so that the binding of the tRNA-Ala to the ACA-Thr codon in mRNA becomes possible.
In analyzing the function of all suppressors, it is important to note that in addition to suppressing a mutation, they are also competing with the normal tRNAs or termination factors for the wildtype codons, and overly successful competition would be an undesirable consequence. In the case of nonsense suppressors, this is dealt with in two ways. (1) The efficiency with which a stop
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codon is read is a function of context – in particular, the sequence 3

to the mutant codon has a strong effect. Thus nonsense codons which do not appear in the normal context of stop codons, are more efficiently read by the suppressors than true termination codons appearing at proper locations. The suppressors are also present in low concentration, which makes them relatively inefficient competitors with wild-type tRNAs or termination factors for wild type codons. Hence, read-through of true termination codons will be minimized. In the case of missense suppressors, low concentration of mutant tRNAs is the only protective strategy, and probably as a result, there are no strong missense suppressors.
Several frameshift suppressors have also been identified. A general strategy for +1 frameshifts is correction by virtue of tRNAs with a 4-base anticodon. The mutation is an insertion of an extra base into the anticodon loop. Thus, the suppressor tRNA
Gly
, which has the anticodon CCCC← corrects a common frame-shift occurring in a run of Gs. The tRNA
Lys
frame shift suppressor responds to either AAAA or AAAU (rather than the w.t. AAA). Yet another suppressor recognizes any 4-base sequence having ACC in the first three positions. In this case, the indiscriminate pairing of the 4 th
position in the anticodon is not determined by the Wobble rules, but an extra base 5′ to the anticodon in the anticodon stem of the tRNA, which creates steric hindrance at the 3

nucleotide of the codon and forces the incoming tRNA to recognize a reading frame that has slipped one position.
In bacteria, -1 frameshift suppressors have also been characterized: E. coli genes hopR1 and hopEl code for tRNAs that insert a single amino acid, valine, for the five-nucleotide sequence
GUGUA. These results were interpreted to be due to detachment of the tRNA from underlined
GUG but retention of the tRNA within the ribosome while the message slipped forward by two bases (instead of three) with the effect of repairing the tRNA to the over lined GUA triplet.
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