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Mechanisms of Gene Mutation

16. Mechanisms of Gene Mutation

Key Concepts

Mutations can occur spontaneously owing to several different mechanisms, including

errors of DNA replication and spontaneous damage to the DNA.

Mutagens are agents that increase the frequency of mutagenesis, usually by altering

the DNA.

Potentially mutagenic and carcinogenic compounds can be detected easily by

mutagenesis tests with bacterial systems.

Biological repair systems eliminate many potentially mutagenic alterations in theDNA.

Cells lacking certain repair systems have higher than normal mutation rates.

Introduction

This chapter describes the main types of molecular processes that give rise to the

mutant alleles discussed in the previous chapter. Such processes are relevant not only

to experimental genetics but also have direct bearing on human health.

Molecular basis of gene mutations

Gene mutations can arise spontaneously or they can be induced. Spontaneous

mutationsare naturally occurring mutations and arise in all cells. Induced mutations

are produced when an organism is exposed to a mutagenic agent, or mutagen; such

mutations typically occur at much higher frequencies than spontaneous mutations do.

To understand the mechanisms of gene mutation requires analysis at the level of DNA

and protein molecules. Preceding chapters have described models for DNA and

protein structure and have considered the nature of mutations that alter these

structures (see, for instance, Table 15-1).

Molecular genetics techniques can be used to determine the sequence of large

segments of DNA and the sequence changes resulting from mutations. Sequencing

has greatly increased our understanding of the pathways that lead to mutagenesis and

has even helped to unravel the mysteries of mutational hot spotsgenetic sites with a

penchant for mutating.

Much work on the molecular basis of mutation has been carried out in single-celled

bacteria and their viruses. However, many mutations leading to inherited diseases in

humans also have been analyzed. We shall review some of the findings of these

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studies. We shall also consider biological repair mechanisms, because repair systems

play a key role in mutagenesis, operating to lower the final observed mutation rates.

For example, inEscherichia coli,with all repair systems functioning, base

substitutions occur at rates of 1010to 109per base pair per cell per generation. As a

general principle, base substitutions arise by a perturbation of the normal pairing ofcomplementary bases.

Spontaneous mutations

Spontaneous mutations arise from a variety of sources, including errors in DNA

replication, spontaneous lesions, and transposable genetic elements. The first two are

considered in this section; the third is examined in Chapter 20.

Errors in DNA replication

An error in DNA replication can occur when an illegitimate nucleotide pair (say, AC)forms in DNA synthesis, leading to a base substitution.

Each of the bases in DNA can appear in one of several forms, called tautomers,

which are isomers that differ in the positions of their atoms and in the bonds between

the atoms. The forms are in equilibrium. The ketoform of each base is normally

present in DNA (Figure 16-1), whereas the iminoand enolforms of the bases are rare.

The ability of the wrong tautomer of one of the standard bases to mispair and cause a

mutation in the course of DNA replication was first noted by Watson and Crick when

they formulated their model for the structure of DNA (Chapter 8). Figure 16-2

demonstrates some possible mispairs resulting from the change of one tautomer intoanother, termed a tautomeric shift.

Mispairs can also result when one of the bases becomes ionized.This type of mispair

may occur more frequently than mispairs due to imino and enol forms of bases.

Transitions.

All the mispairs described so far lead to transition mutations,in which a purine

substitutes for a purine or a pyrimidine for a pyrimidine (Figure 16-3). The bacterial

DNA polymerase III (Chapter 8) has an editing capacity that recognizes such

mismatches and excises them, thus greatly reducing the observed mutations. Another

repair system (described later in this chapter) corrects many of the mismatched bases

that escape correction by the polymerase editing function.

Transversions.

In transversion mutations,a pyrimidine substitutes for a purine or vice versa.

Transversions cannot be generated by the mismatches depicted in Figure 16-2. With

bases in the DNA in the normal orientation, creation of a transversion by a replication

error would require, at some point in the course of replication, mispairing of a purine

with a purine or a pyrimidine with a pyrimidine. Although the dimensions of the DNA

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double helix render such mispairs energetically unfavorable, we now know from

X-ray diffraction studies that GA pairs, as well as other purinepurine pairs, can

form.

Frameshift mutations.

Replication errors can also lead to frameshift mutations.Recall from Chapter 10that

such mutations result in greatly altered proteins.

In the mid-1960s, George Streisinger and his coworkers deduced the nucleotide

sequence surrounding different sites of frameshift mutations in the lysozyme gene of

phage T4. They found that these mutations often occurred at repeated sequences and

formulated a model to account for frameshifts in DNA synthesis. In the Streisinger

model (Figure 16-4), frameshifts arise when loops in single-stranded regions are

stabilized by the slipped mispairing of repeated sequences.

In the 1970s, Jeffrey Miller and his co-workers examined mutational hot spots in the

lacIgene ofE. coli.As already mentioned, hot spots are sites in a gene that are much

more mutable than other sites. The lacIwork showed that certain hot spots result from

repeated sequences, just as predicted by the Streisinger model. Figure 16-5depicts the

distribution of spontaneous mutations in the lacIgene. Compare this distribution with

that in the rIIgenes of T4 seen by Benzer (see Figure 9-26). Note how one or two

mutational sites dominate the distribution in both cases. In lacI,a four-base-pair

sequence repeated three times in tandem in the wild type is the cause of the hot spots

(for simplicity, only one strand of the double strand of DNA is indicated):

The major hot spot, represented here by the mutationsFS5, FS25, FS45,andFS65,

results from the addition of one extra set of the four bases CTGG to one strand of the

DNA. This hot spot reverts at a high rate, losing the extra set of four bases. The minor

hot spot, represented here by the mutationsFS2andFS84,results from the loss of one

set of the four bases CTGG. This mutant does not readily regain the lost set of four

base pairs.

How can the Streisinger model explain these observations? The model predicts that

the frequency of a particular frameshift depends on the number of base pairs that can

form during the slipped mispairing of repeated sequences. The wild-type sequence

shown for the lacIgene can slip out one CTGG sequence and stabilize structure by

forming nine base pairs. (Can you work this out by applying the model in Figure 16-4

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to the sequence shown for lacI?) Whether a deletion or an addition is generated

depends on whether the slippage occurs on the template or on the newly synthesized

strand, respectively. In a similar fashion, the addition mutant can slip out one CTGG

sequence and stabilize a structure with 13 base pairs (verify this for theFS5sequence

shown for lacI), which explains the rapid reversion of mutations such asFS5.However, there are only five base pairs available to stabilize a slipped-out CTGG in

the deletion mutant, accounting for the infrequent reversion of mutations such as FS2

in the sequence shown for lacI.

Deletions and duplications.

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Large deletions(more than a few base pairs) constitute a sizable fraction of

sponta-neous mutations, as shown in Figure 16-5. The majority, although not all, of

the deletions occur at repeated sequences. Figure 16-6shows the results for the first

12 deletions analyzed at the DNA sequence level, presented by Miller and his

co-workers in 1978. Further studies showed that hot spots for deletions are in the

longest repeated sequences. Duplicationsof segments of DNA have been observed in

many organisms. Like deletions, they often occur at sequence repeats.

How do deletions and duplications form? Several mechanisms could account for their

formation. Deletions may be generated as replication errors. For example, an

extension of the Streisinger model of slipped mispairing (Figure 16-4) could explain

why deletions predominate at short repeated sequences. Alternatively, deletions and

duplications could be generated by recombinational mechanisms (to be described in

Chapter 19).Spontaneous lesions

In addition to replication errors, spontaneous lesions,naturally occurring damage to

the DNA, can generate mutations. Two of the most frequent spontaneous lesions

result from depurination and deamination.

Depurination,the more common of the two, consists of the interruption of the

glycosidic bond between the base and deoxyribose and the subsequent loss of a

guanine or an adenine residue from the DNA (Figure 16-7). A mammalian cell

spontaneously loses about 10,000 purines from its DNA in a 20-hour cell-generationperiod at 37C. If these lesions were to persist, they would result in significant genetic

damage because, in replication, the resulting apurinic sitescannot specify a base

complementary to the original purine. However, as we shall see later in the chapter,

efficient repair systems remove apurinic sites. Under certain conditions (to be

described later), a base can be inserted across from an apurinic site; this insertion will

frequently result in a mutation.

The deaminationof cytosine yields uracil (Figure 16-8a). Unrepaired uracil residues

will pair with adenine in replication, resulting in the conversion of a GC pair into an

AT pair (a GCAT transition). In 1978, deaminations at certain cytosine residues

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were found to be the cause of one type of mutational hot spot. DNA sequence analysis

of GC AT transition hot spots in the lacIgene showed that 5-methylcytosine

residues are pres-ent at each hot spot. (Certain bases in prokaryotes and eukaryotes

are methylated.) Some of the data from this lacIstudy are shown in Figure 16-9. The

height of each bar on the graph represents the frequency of mutations at each of anumber of sites. It can be seen that the positions of 5-methylcytosine residues

correlate nicely with the most mutable sites.

Why are 5-methylcytosines hot spots for mutations? One of the repair enzymes in the

cell, uracil-DNA glycosylase, recognizes the uracil residues in the DNA that arise

from deaminations and excises them, leaving a gap that is subsequently filled (a

process to be described later in the chapter). However, the deamination of

5-methylcytosine (Figure 16-8b) generates thymine (5-methyluracil), which is not

recognized by the enzyme uracil-DNA glycosylase and thus is not repaired. Therefore,

C

T transitions generated by deamination are seen more frequently at5-methylcytosine sites, because they escape this repair system.

A consequence of the frequent mutation of 5-methylcytosine to thymine is the

underrepresentation of CpG dinucleotides in higher cells, because this sequence is

methylated to give 5-methyl-CpG, which is gradually converted into TpG.

Oxidatively damaged basesrepresent a third type of spontaneous lesion implicated

in mutagenesis. Active oxygen species, such as superoxide radicals (O2), hydrogen

peroxide (H2O2), and hydroxyl radicals (OH), are produced as by-products of normal

aerobic metabolism. They can cause oxidative damage to DNA, as well as to

precursors of DNA (such as GTP), which results in mutation and which has been

implicated in a number of human diseases. Figure 16-10shows two products of

oxidative damage. The 8-oxo-7-hydrodeoxyguanosine (8-oxodG, or GO) product

frequently mispairs with A, resulting in a high level of G T transversions.

Thymidine glycol blocks DNA replication if unrepaired but has not yet been

implicated in mutagenesis.

MESSAGE

Spontaneous mutations can be generated by different processes. Replication errorsand spontaneous lesions generate most of the base-substitution and frameshift

mutations. Replication errors may also cause some deletions that occur in the

absence of mutagenic treatment.

Spontaneous mutations and human diseases

DNA sequence analysis has revealed the mutations responsible for a number of

human hereditary diseases. The previously discussed studies of bacterial mutations

allow us to suggest mechanisms that cause these human disorders.

A number of these disorders are due to deletionsor duplicationsinvolving repeated

sequences. For example, mitochondrial encephalomyopathies are a group of disorders

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affecting the central nervous system or the muscles (Kearns-Sayre syndrome). They

are characterized by dysfunction of oxidation phosphorylation (a function of the

mitochondria) and by changes in mitochondrial structure. These disorders have been

shown to result from deletions that occur between repeated sequences. Figure 16-11

depicts one of these deletions. Note how similar it is in form to the spontaneousE.colideletions shown in Figure 16-6.

A common mechanism that is responsible for a number of genetic diseases is the

expansion of a three-base-pair repeat,as in fragile X syndrome (Figure 16-12).

This syndrome is the most common form of inherited mental retardation, occurring in

close to 1 of 1500 males and 1 of 2500 females. It is evidenced cytologically by a

fragile site in the X chromosome that results in a break in vitro.

The inheritance of fragile X syndrome is unusual in that 20 percent of the males with

a fragile X chromosome are phenotypically normal but transmit the affected

chromosome to their daughters, who also appear normal. These males are said to be

normally transmitting males (NTMs). However, the sons of the daughters of the

NTMs frequently display symptoms. The fragile X syndrome results from mutations

in a (CGG)nrepeat in the coding sequence of theFMR-1gene. Patients with the

disease show specific methylation, induced by the mutation, at a nearby CpG cluster,

resulting in reducedFMR-1expression.

Why do symptoms develop in some persons with a fragile X chromosome and not in

others? The answer seems to lie in the number of CGG repeats in theFMR-1gene.

Humans normally show a considerable variation in the number of CGG repeats in the

FMR-1gene, ranging from 6 to 54, with 29 repeats in the most frequent allele. [The

variation in CGG repeats produces a corresponding variation in the number of

arginine residues (CGG is an arginine codon) in theFMR-1-encoded protein.] Both

NTMs and their daughters have a much larger number of repeats, ranging from 50 to

200. These increased repeats have been termed premutations.All premutation alleles

are unstable. The males and females with symptoms of the disease, as well as many

carrier females, have additional insertions of DNA, suggesting repeat numbers of 200

to 1300. The frequency of expansion has been shown to increase with the size of the

DNA insertion (and thus, presumably, with the number of repeats). Apparently, the

number of repeats in the premutation alleles found in NTMs and their daughters isabove a certain threshold and thus is much more likely to expand to a full mutation

than is the case for normal persons.

The proposed mechanism for these repeats is a slipped mispairing in DNA synthesis

(as shown in Figure 16-6) involving a one-step expansion of the four-base-pair

sequence CTGG. However, the extraordinarily high frequency of mutation at the

three-base-pair repeats in the fragile X syndrome suggests that in human cells, after a

threshold level of about 50 repeats, the replication machinery cannot faithfully

replicate the correct sequence, and large variations in repeat numbers result.

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A second inherited disease, X-linked spinal and bulbar muscular atrophy (known as

Kennedy disease), also results from the amplification of a three-base-pair repeat, in

this case a repeat of the CAG triplet. Kennedy disease, which is characterized by

progressive muscle weakness and atrophy, results from mutations in the gene that

encodes the androgen receptor. Normal persons have an average of 21 CAG repeats inthis gene, whereas affected patients have repeats ranging from 40 to 52.

Myotonic dystrophy, the most common form of adult muscular dystrophy, is yet

another example of sequence expansion causing a human disease. Susceptible

families display an increase in severity of the disease in successive generations; this

increase is caused by the progressive amplification of a CTG triplet at the 3end of a

transcript. Normal people possess, on average, five copies of the CTG repeat; mildly

affected people have approximately 50 copies, and severely affected people have

more than 1000 repeats of the CTG triplet. Additional examples of triplet expansion

are still appearingfor instance, Huntington disease.

Figure 16-1. Pairing between the normal (keto) forms of the bases.

Figure 16-2. Mismatched bases. (a) Mispairs resulting from rare tautomeric forms of

the pyrimidines; (b) mispairs resulting from rare tautomeric forms of the purines.



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Figure 16-3. Mutation by tautomeric shifts in the bases of DNA. (a) In the examplediagrammed, a guanine undergoes a tautomeric shift to its rare enol form (G*) at the

time of replication. (b) In its enol form, it pairs with thymine. (c and d) In the next

replication, the guanine shifts back to its more stable keto form. The thymine

incorporated opposite the enol form of guanine, seen in part b, directs the

incorporation of adenine in the subsequent replication, shown in parts c and d. The net

result is a GC AT mutation. If a guanine undergoes a tautomeric shift from the

common keto form to the rare enol form at the time of incorporation (as a nucleoside

triphosphate, rather than in the template strand diagrammed here), it will be

incorporated opposite thymine in the template strand and cause an AT GC

mutation. (From E. J. Gardner and D. P. Snustad,Principles of Genetics,5th ed.

Copyright 1984 by John Wiley & Sons, New York.)

Figure 16-4. A simplified version of the Streisinger model for frameshift formation.

(ac) In DNA synthesis, the newly synthesized strand slips, looping out one or several

bases. This loop is stabilized by the pairing afforded by the repetitive-sequence unit

(the A bases in this case). An addition of one base pair, AT, will result at the next

round of replication in this example. (df) If, instead of the newly synthesized strand,

the template strand slips, then a deletion results. Here the repeating unit is a CT



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dinucleotide. After slippage, a deletion of two base pairs (CG and TA) would result

at the next round of replication.

The Streisinger Model.

Figure 16-5. The distribution of 140 spontaneous mutations in lacI.Each occurrence

of a point mutation is indicated by a box. Red boxes designate fast-reverting

mutations. Deletions (gold) are represented below. TheImap is given in terms of the

amino acid number in the correspondingI-encoded lacrepressor. Allele numbers refer

to mutations that have been analyzed at the DNA sequence level. The mutations S114

and S58(circles) result from the insertion of transposable elements (see Chapter 20).

S28(red circle) is a duplication of 88 base pairs. (From P. J. Farabaugh, U.

Schmeissner, M. Hofer, and J. H. Miller,Journal of Molecular Biology126, 1978,

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847.)

Figure 16-6. Deletions in lacI.Deletions occurring in S74and S112are shown at the

top of the figure. As indicated by the gold bars, one of the sequence repeats (aqua)and all the intervening DNA are deleted, leaving one copy of the repeated sequence.

All mutations were analyzed by direct DNA sequence determination. (From P. J.

Farabaugh, U. Schmeissner, M. Hofer, and J. H. Miller,Journal of Molecular Biology

126, 1978, 847.)

Figure 16-7. The loss of a purine residue (guanine) from a single strand of DNA. The

sugar-phosphate backbone is left intact.



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Figure 16-8. Deamination of (a) cytosine and (b) 5-methylcytosine.

Figure 16-9. 5-Methylcytosine hot spots inE. coli.Nonsense mutations occurring at

15 different sites in lacIwere scored. All result from the GC AT transition. The

asterisks (*) mark the positions of 5-methylcytosines. Open bars depict sites at which

the GC AT change could be detected but at which no mutations occurred in this

particular collection. It can be seen that 5-methylcytosine residues are hot spots forthe GC AT transition. Of 50 independently occurring mutations, 44 were at the 4

methylated cytosine sites and only 6 were at the 11 unmethylated cytosines. (From C.

Coulondre et al.,Nature274, 1978, 775.).



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Figure 16-10. DNA damage products formed after attack by oxygen radicals. dR =

deoxyribose.

Figure 16-11. Sequences of wild-type (WT) mitochondrial DNA and deleted DNA

(KS) from a patient with Kearns-Sayre syndrome. The 13-base boxed sequence is

identical in both WT and KS and serves as a breakpoint for the DNA deletion. Asingle base (boldface type) is altered in KS, aside from the deleted segment.

Figure 16-12. Expansion of the CGG triplet in theFMR-1gene seen in the fragile X

syndrome. Normal persons have from 6 to 54 copies of the CGG repeat, whereas

members of susceptible families display an increase (premutation) in the number of

repeats: normally transmitting males (NTMs) and their daughters are phenotypically

normal but display from 50 to 200 copies of the CGG triplet; the number of repeats

expands to some 200 to 1300 in those showing full symptoms of the disease.

Induced mutations

Mutational specificity



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When we observe the distribution of mutations induced by different mutagens, we see

a distinct specificity that is characteristic of each mutagen. Such mutational

specificitywas first noted in the phage T4 rIIsystem by Benzer in 1961. Specificity

arises from a given mutagen's preference both for a certain typeof mutation (for

example, GC

AT transitions) and for certain mutationalsites(hot spots). Figure16-13shows the mutational specificity in lacIof three mutagens described later:

ethylmethanesulfonate (EMS), ultraviolet (UV) light, and aflatoxin B1(AFB1). The

graphs show the distribution of base-substitution mutations that create

chain-terminating UAG codons. Figure 16-13is similar to Figure 9-26, which shows

the distribution of mutations in rII, except that the specific sequence changes are

known for each lacIsite, allowing the graphs to be broken down into each category of

substitution.

Figure 16-13reveals the two components of mutational specificity. First, each

mutagen shown favors a specific category of substitution. For example, EMS and UVfavor GC AT transitions, whereas AFB1favors GC TA transversions. These

preferences are related to the different mechanisms of mutagenesis. Second, even

within the same category, there are large differences in mutation rate. These

differences can be seen best with UV light for the GC AT changes. Some aspect of

the surrounding DNA sequence must cause these differences. In some cases, the cause

of mutational hot spots can be determined by DNA sequence studies, as previously

described for 5-methylcytosine residues and for certain frameshift sites (Figures 16-5

and 16-9). In many examples of mutagen-induced hot spots, the precise reason for the

high mutability of specific sites is still unknown. However, high lesion frequency at

some sites and reduced repair at certain sites are sometimes causes of hot spots.

Mechanisms of mutagenesis

Mutagens induce mutations by at least three different mechanisms. They can replace a

base in the DNA, alter a base so that it specifically mispairs with another base, or

damage a base so that it can no longer pair with any base under normal conditions.

Incorporation of base analogs.

Some chemical compounds are sufficiently similar to the normal nitrogen bases of

DNA that they occasionally are incorporated into DNA in place of normal bases; such

compounds are called base analogs.Once in place, these analogs have pairing

properties unlike those of the normal bases; thus, they can produce mutations by

causing incorrect nucleotides to be inserted opposite them in replication. The original

base analog exists in only a single strand, but it can cause a nucleotide-pair

substitution that is replicated in all DNA copies descended from the original strand.

For example, 5-bromouracil (5-BU)is an analog of thymine that has bromine at the

C-5 position in place of the CH3group found in thymine. This change does not affect

the atoms that take part in hydrogen bonding in base pairing, but the presence of thebromine significantly alters the distribution of electrons in the base. The normal

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structure (the keto form) of 5-BU pairs with adenine, as shown in Figure 16-14a.

5-BU can frequently change to either the enol form or an ionized form; the latter pairs

in vivo with guanine (Figure 16-14b). Thus, the nature of the pair formed in

replication will depend on the form of 5-BU at the moment of pairing (Figure 16-15).

5-BU causes transitions almost exclusively, as predicted in Figures 16-14and 16-15.

Another analog widely used in research is 2-amino-purine (2-AP),which is an

analog of adenine that can pair with thymine but can also mispair with cytosine when

protonated, as shown in Figure 16-16. Therefore, when 2-AP is incorporated into

DNA by pairing with thymine, it can generate AT GC transitions by mispairing

with cytosine in subsequent replications. Or, if 2-AP is incorporated by mispairing

with cytosine, then GC AT transitions will result when it pairs with thymine.

Genetic studies have shown that 2-AP, like 5-BU, is very specific for transitions.

Specific mispairing.

Some mutagens are not incorporated into the DNA but instead alter a base, causing

specific mispairing. Certain alkylating agents,such as ethylmethanesulfonate

(EMS)and the widely used nitrosoguanidine (NG),operate by this pathway:

Although such agents add alkyl groups (an ethyl group in EMS and a methyl group in

NG) to many positions on all four bases, mutagenicity is best correlated with an

addition to the oxygen at the 6 position of guanine to create an O-6-alkylguanine. This

addition leads to direct mispairing with thymine, as shown in Figure 16-17, and would

result in GC AT transitions at the next round of replication. As expected,

determinations of mutagenic specificity for EMS and NG show a strong preferencefor GC AT transitions (see the data for EMS shown in Figure 16-13). Alkylating

agents can also modify the bases in dNTPs (where N is any base), which are

precursors in DNA synthesis.

The intercalating agentsform another important class of DNA modifiers. This group

of compounds includes proflavin, acridine orange,and a class of chemicals termed

ICR compounds(Figure 16-18a). These agents are planar molecules, which mimic

base pairs and are able to slip themselves in (intercalate)between the stacked

nitrogen bases at the core of the DNA double helix (Figure 16-18b). In this

intercalated position, the agent can cause single-nucleotide-pair insertions or deletions.

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Intercalating agents may also stack between bases in single-stranded DNA; in so

doing, they may stabilize bases that are looped out during frameshift formation, as

depicted in the Streisinger model (Figure 16-4).

Base damage.

A large number of mutagens damage one or more bases, so no specific base pairing is

possible. The result is a replication block, because DNA synthesis will not proceed

past a base that cannot specify its complementary partner by hydrogen bonding. In

bacterial cells, such replication blocks can be bypassedby inserting nonspecific bases.

The process requires the activation of a special system, the SOS system(Figure

16-19). The name SOS comes from the idea that this system is induced as an

emergency response to prevent cell death in the presence of significant DNA damage.

SOS induction is a last resort, allowing the cell to trade death for a certain level of

mutagenesis.

Exactly how the SOS bypass system functions is not clear, although in E. coliit is

known to be dependent on at least three genes, recA(which also has a role in general

recombination), umuC,and umuD.Current models for SOS bypass suggest that the

UmuC and UmuD proteins combine with the polymerase III DNA replication

complex to loosen its otherwise strict specificity and permit replication past

noncoding lesions.

Figure 16-19shows a model for the bypass system operating after DNA polymerase

III stalls at a type of damage called a TC photodimer. Because replication can restart

downstream from the dimer, a single-stranded region of DNA is generated. This

region attracts the stabilizing protein, called single-strand-binding protein (Ssb), as

well as the RecA protein, which forms filaments and signals the cell to synthesize the

UmuC and UmuD proteins. The UmuD protein binds to the filaments and is cleaved

by the RecA protein to yield a shortened version termed UmuD, which then recruits

the UmuC protein to form a complex that allows DNA polymerization to continue

past the dimer, adding bases across from the dimer with a high error frequency (see

Figure 16-19).

Therefore mutagens that damage specific base-pairing sites are dependent on the SOS

system for their action. The category of SOS-dependent mutagens is important,

because it includes most cancer-causing agents (carcinogens), such as ultraviolet light,

aflatoxin B1, and benzo(a)pyrene (discussed later).

How does the SOS system take part in the recovery of mutations after mutagenesis?

Does the SOS system lower the fidelity of DNA replications so much (to permit the

bypass of noncoding lesions) that many replication errors occur, even for undamaged

DNA? If this hypothesis were correct, most mutations generated by different

SOS-dependent mutagens would be similar, rather than specific to each mutagen.

Most mutations would result from the action of the SOS system itself on undamaged

DNA. The mutagen, then, would play the indirect role of inducing the SOS system.

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Studies of mutational specificity, however, have shown that this is not the case.

Instead, a series of different SOS-dependent mutagens have markedly different

specificities, as seen for UV light and aflatoxin B1in Figure 16-13. Each mutagen

induces a unique distribution of mutations. Therefore, the mutations must be

generated in response to specific damaged base pairs. The type of lesion differs inmany cases. Some of the most widely studied lesions include UV photoproducts and

apurinic sites.

Ultraviolet lightgenerates a number of photoproducts in DNA. Two different lesions

that occur at adjacent pyrimidine residuesthe cyclobutane pyrimidine photodimer

and the 6-4 photoproduct (Figure 16-20)have been most strongly correlated with

mutagenesis. These lesions interfere with normal base pairing; hence, induction of the

SOS system is required for mutagenesis. The insertion of incorrect bases across from

UV photoproducts is at the 3position of the dimer, and more frequently for 5-CC-3

and 5-TC-3

dimers. The C

T transition is the most frequent mutation, but other

base substitutions (transversions) and frameshifts also are stimulated by UV light, as

are duplications and deletions. The mutagenic specificity of UV light is illustrated in

Figure 16-13.

Ionizing radiation

Ionizing radiation results in the formation of ionized and excited molecules that can

cause damage to cellular components and to DNA. Because of the aqueous nature of

biological systems, the molecules generated by the effects of ionizing radiation on

water produce the most damage. Many different types of reactive oxygen specials areproduced, including superoxide radicals, such as OH. The most biologically relevant

reaction products are OH, O2, and H2O2. These species can damage bases and cause

different adducts and degradation products. Among the most prevalent, which result

in mutations, are thymine glycol and 8-oxodG, pictured in Figure 16-10. Ionizing

radiation can cause breakage of the N-glycosydic bond, leading to the formation of

AP sites, and can cause strand breaks that are responsible for most of the lethal effects

of such radiation.

Aflatoxin B1is a powerful carcinogen. It generates apurinic sitesfollowing the

formation of an addition product at the N-7 position of guanine (Figure 16-21).Studies with apurinic sites generated in vitro demonstrated a requirement for the SOS

system and showed that the SOS bypass of these sites leads to the preferential

insertion of an adenine across from an apurinic site. Thus agents that cause

depurination at guanine residues should preferentially induce GC TA transversions.

Can you see why the insertion of an adenine across from an apurinic site derived from

a guanine would generate this substitution at the next round of replication? Figure

16-13shows the genetic analysis of many base substitutions induced by AFB1. You

can verify that most of the substitutions are indeed GC TA transversions.



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AFB1is a member of a class of chemical carcinogens known as bulky addition

productswhen they bind covalently to DNA. Other examples include the diol

epoxides of benzo(a)pyrene,a compound produced by internal combustion engines.

For many different compounds, it is not yet clear which DNA addition products play

the principal role in mutagenesis. In some cases, the mutagenic specificity suggeststhat depurination may be an intermediate step in mutagenesis; in others, the question

of which mechanism is operating is completely open.

MESSAGE

Mutagens induce mutations by a variety of mechanisms. Some mutagens mimicnormal bases and are incorporated into DNA, where they can mispair. Others

damage bases and either cause specific mispairing or destroy pairing by causing

nonrecognition of bases. In the latter case, a bypass system, the SOS system, must be

induced to allow replication past the lesion.



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Figure 16-13. Specificity of mutagens. The distribution of mutations among 36 sites

in the lacIgene is shown for three mutagens: EMS, UV light, and aflatoxin B1. The

height of each bar represents the number of occurrences of mutations at the respective

site. Some hot spots are shown off-scale, with the number of occurrences indicated

directly above the respective peak. For instance, in the UV-generated collection, onesite resulting from a GC AT transition is represented by 80 occurrences. Each

mutational site represented in the figure generates an amber (UAG) codon in the

corresponding mRNA. The mutations are arranged according to the type of base

substitution. Asterisks mark the positions of 5-methylcytosines. (After C. Coulondre

and J. H. Miller,Journal of Molecular Biology117, 1977, 577; and P. L. Foster et al.,

Proceedings of the National Academy of Sciences USA80, 1983, 2695.)

Figure 16-14. Alternative pairing possibilities for 5-bromouracil (5-BU). 5-BU is an

analog of thymine that can be mistakenly incorporated into DNA as a base. It has a

bromine atom in place of the methyl group. (a) In its normal keto state, 5-BU mimics

the pairing behavior of the thymine that it replaces, pairing with adenine. (b) The

presence of the bromine atom, however, causes a relatively frequent redistribution of

electrons, so that 5-BU can spend part of its existence in the rare ionized form. In this

state, it pairs with guanine, mimicking the behavior of cytosine and thus inducing

mutations in replication.



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Figure 16-15. The mechanism of 5-BU mutagenesis. 5-BU causes mutations when it

is incorporated in one form and then shifts to another form. (a) In its normal keto state,5-BU pairs like thymine (5-BUT). Thus, 5-BU is incorporated across from adenine

and subsequently mispairs with guanine, resulting in AT GC transitions. (b) In its

ionized form, 5-BU pairs like cytosine (5-BUC). Thus, 5-BU is misincorporated across

from guanine and subsequently pairs with adenine, resulting in GC AT transitions.



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Figure 16-16. Alternative pairing possibilities for 2-aminopurine (2-AP), an analog of

adenine. Normally, 2-AP pairs with thymine (a), but in its protonated state it can pair

with cytosine (b).

Figure 16-17. Alkylation-induced specific mispairing. The alkylation (in this case,

EMS-generated ethylation) of the O-6 position of guanine and the O-4 position of

thymine can lead to direct mispairing with thymine and guanine, respectively, as

shown here. In bacteria, where mutations have been analyzed in great detail, the

principal mutations detected are GC AT transitions, indicating that the O-6

alkylation of guanine is most relevant to mutagenesis.



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Figure 16-18. Intercalating agents. (a) Structures of the common agents proflavin,

acridine orange, and ICR-191. (b) An intercalating agent slips between the

nitrogenous bases stacked at the center of the DNA molecule. This occurrence can

lead to single-nucleotide-pair insertions and deletions. (From L. S. Lerman,Proceedings of the National Academy of Sciences USA49, 1963, 94.)

Figure 16-19. The SOS system. DNA polymerase III, shown in blue, stops at a

noncoding lesion, such as the TC photodimer shown here, generating single-stranded

regions that attract the Ssb protein (dark purple) and RecA (light purple), which forms

filaments. The presence of RecA filaments helps to signal the cell to synthesizeUmuD (red circles), which is cleaved by RecA to yield UmuD (pink circles) and

UmuC (yellow ovals). The UmuC is recruited to form a complex with UmuDthat

permits DNA polymerization to proceed past the blocking lesion.



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Figure 16-20. (a) Structure of a cyclobutane pyrimidine dimer. Ultraviolet light

stimulates the formation of a four-membered cyclobutane ring (green) between two

adjacent pyrimidines on the same DNA strand by acting on the 5,6 double bonds. (b)

Structure of the 6-4 photoproduct. The structure forms most prevalently with 5 -CC-3

and 5-TC-3, between the C-6 and the C-4 positions of two adjacent pyrimidines,

causing a significant perturbation in local structure of the double helix. (Part a after E.

C. Friedberg,DNA Repair.Copyright 1985 by W. H. Freeman and Company; part

b after E. C. Friedberg,DNA Repair.Copyright 1985 by W. H. Freeman and

Company. As adapted from A. M. R. Taylor, C. M. Rosney, and J. B. Campbell,

Cancer Research39, 1979, 10461050.

Figure 16-21. The binding of metabolically activated aflatoxin B1to DNA.



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Reversion analysis

Testing for the reversion of a mutation can tell us something about the nature of the

mutation or the action of a mutagen. For example, if a mutation cannot be reverted by

action of the mutagen that induced it, then the mutagen must have some relativelyspecific unidirectional action. In a mutation induced by hydroxylamine (HA), for

instance, it would be reasonable to expect that the original mutation is GC AT,

which cannot be reverted by another specific GC AT event. Similarly, mutations

that can be reverted by proflavin are in all likelihood frameshift mutations; thus

mutations induced by nitrous acid (NA), which are transitions, should not be

revertible by proflavin.

Transversions cannot be induced by the aforementioned agents, but they are definitely

known to be common among spontaneous mutations, as shown by studies of DNA

and protein sequencing. Thus, in the reversion test, if a mutation revertsspontaneously but does not revert in response to a transition mutagen or a frameshift

mutagen, then, by elimination, it is probably a transversion.

Table 16-1summarizes some reversion expectations based on simple assumptions

from reversion analysis. Recall that mutagen specificities depend on the organism, the

genotype, the gene studied, and the action of biological repair systems. Note that the

kinds of logic employed in the reversion test rely heavily on the assumption that the

reversion events are not due to suppressors or transposable elements; either of them

would make inference from reversion more difficult.

Table 16-1. Reversion Tests

REVERSION MUTAGENS

Mutation NA HA or EMS Proflavin Spontaneous reversion

Transition (GC AT) + +

Transition (AT GC) + + +

Transversion +

Frameshift + +

Note:A plus sign indicates a measurable rate of reversion due to a given mutagen.NA, nitrousacid; HA, hydroxylamine; EMS, ethyl methanesulfonate.

Relation between mutagens and carcinogens

Mutagenicity and carcinogenicity are clearly correlated. One study showed that 157 of

175 known carcinogens (approximately 90 percent) are also mutagens. The somatic

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mutation theoryof cancer holds that these agents cause cancer by inducing the

mutation of somatic cells. Thus, understanding mutagenesis is of great relevance to

our society.

Induced mutations and human cancer

Understanding the specificity of mutagens in bacteria has led to the direct implication

of certain environmental mutagens in the causation of human cancers. Ultraviolet

light and aflatoxin B1have long been suspected of causing skin cancer and liver

cancer, respectively. Now, DNA sequence analysis of mutations in a human cancer

gene has provided direct evidence of their involvement. The gene in question is

termedp53and is one of a number of tumor-suppressor genesgenes that encode

proteins that suppress tumor formation. (We will learn more about these genes in

Chapter 23.) A sizable proportion of human cancer patients have mutated

tumor-suppressor genes.

Liver cancer is prevalent in southern Africa and East Asia, and a high exposure to

AFB1in these regions has been correlated with the high incidence of liver cancer.

Whenp53mutations in cancer patients were analyzed, G T transversions, the

signature of AFB1-induced mutations, were found in liver cancer patients from South

Africa and East Asia but not in patients from these regions with lung, colon, or breast

cancer. On the other hand,p53mutations in liver cancer patients from areas of low

AFB1exposure did not result from G T transversions. These findings, together

with the results from the mutagenic specificity studies of AFB1(see Figure 16-13),

allow us to conclude that AFB1-induced mutations are a prime cause of liver cancer inSouth Africa and East Asia.

Sequencingp53mutations has also strengthened the link between UV and human skin

cancers. The majority of invasive human squamous cell carcinomas analyzed so far

havep53mutations, all of them mutations at dipyrimidine sites, most of which are C

T substitutions when the C is the 3 pyrimidine of a TC dimer. This is the profile of

UV-induced mutations. In addition, several tumors havep53mutations resulting from

a CC TT double base change, which is found most frequently among UV-induced

mutations.

The modern environment exposes everyone to a wide variety of chemicals in drugs,

cosmetics, food preservatives, pesticides, compounds used in industry, pollutants, and

so forth. Many of these compounds have been shown to be carcinogenic and

mutagenic. Examples include the food preservative AF-2, the food fumigant ethylene

dibromide, the antischistosome drug hycanthone, several hair-dye additives, and the

industrial compound vinyl chloride; all are potent, and some have subsequently been

subjected to government control. However, hundreds of new chemicals and products

appear on the market each week. How can such vast numbers of new agents be tested

for carcinogenicity before much of the population has been exposed to them?

Ames test

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Many test systems have been devised to screen for carcinogenicity. These tests are

time consuming, typically requiring laborious research with small mammals. More

rapid tests do exist that make use of microbes (such as fungi or bacteria) and test for

mutagenicity rather than carcinogenicity. The most widely used test was developed in

the 1970s by Bruce Ames, who worked with Salmonella typhimurium.This Ames testuses two auxotrophic histidine mutations, which revert by different molecular

mechanisms (Figure 16-22). Further properties were genetically engineered into these

strains to make them suitable for mutagen detection. First, they carry a mutation that

inactivates the excision-repair system (described later). Second, they carry a mutation

that eliminates the protective lipopolysaccharide coating of wild-type Salmonellato

facilitate the entry of many different chemicals into the cell.

Bacteria are evolutionarily a long way removed from humans. Can the results of a test

on bacteria have any real significance in detecting chemicals that are dangerous for

humans? First, we have seen that the genetic and chemical nature of DNA is identicalin all organisms, so a compound acting as a mutagen in one organism is likely to have

some mutagenic effects in other organisms. Second, Ames devised a way to simulate

the human metabolism in the bacterial system. In mammals, much of the important

processing of ingested chemicals takes place in the liver, where externally derived

compounds normally are detoxified or broken down. In some cases, the action of liver

enzymes can create a toxic or mutagenic compound from a substance that was not

originally dangerous (Figure 16-23). Ames incorporated mammalian liver enzymes in

his bacterial test system, using rat livers for this purpose. Figure 16-24outlines the

procedure used in the Ames test.

Chemicals detected by this test can be regarded not only as potential carcinogens

(sources of somatic mutations), but also as possible causes of mutations in germinal

cells. Because the test system is so simple and inexpensive, many laboratories

throughout the world now routinely test large numbers of potentially hazardous

compounds for mutagenicity and potential carcinogenicity.

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Figure 16-22. Ames test results showing the mutagenicity of aflatoxin B1, which is

also a potent carcinogen. TA100, TA1538, and TA1535 are strains of Salmonella

bearing different hisauxotrophic mutations. The TA100 strain is highly sensitive to

reversion through base-pair substitution. The TA1535 and TA1538 strains are

sensitive to reversion through frameshift mutation. The test results show that aflatoxin

B1is a potent mutagen that causes base-pair substitutions but not frameshifts. (From J.

McCann and B. N. Ames, in W. G. Flamm and M. A. Mehlman, eds.,Advances in

Modern Technology,Vol. 5. Copyright by Hemisphere Publishing Corp.,



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Washington, DC.)

Figure 16-23. The metabolic conversion of benzo(a)pyrene (BP) into a mutagen (and

a carcinogen). Benzo(a)pyrene goes through several steps (a) as it is made more water

soluble prior to excretion. One of the intermediates in this process, a diol epoxide (3),

is capable of reacting with guanine in DNA (b). This reaction leads to a distortion of

the DNA molecule (c) and mutations. Benzo(a)pyrene is therefore a mutagen for anycell that has the enzymes that produce this intermediate. (After I. B. Weinstein et al.,

Science193, 1976, 592; from J. Cairns, Cancer: Science and Society.Copyright

1978 by W. H. Freeman and Company.)



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Figure 16-24. Summary of the procedure used for the Ames test. First, rat liver

enzymes are mobilized by injecting the animals with Arochlor. (Enzymes from the

liver are used because they carry out the metabolic processes of detoxifying and

toxifying body chemicals.) The rat liver is then homogenized, and the supernatant of

solubilized liver enzymes (S9) is added to a suspension of auxotrophic bacteria in a

solution of the potential carcinogen (X). This mixture is plated on a medium

containing no histidine, and revertants of mutant strains 1 and 2 are looked for. A

control experiment containing no potential carcinogen is always run simultaneously.

The presence of revertants indicates that the chemical is a mutagen and possibly a

carcinogen as well.

Biological repair mechanisms

As we have seen in the preceding discussions, there are many potential threats to the

fidelity of DNA replication. Not only is there an inherent error rate for the replication

of DNA, but there are also spontaneous lesions that can provoke additional errors.

Moreover, mutagens in the environment can damage DNA and greatly increase the

mutation rate.

Living cells have evolved a series of enzymatic systems that repair DNA damage in a

variety of ways. Failure of these systems can lead to a higher mutation rate. A number



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of human diseases including certain types of cancer can be attributed to defects in

DNA repair, as we shall see later. Let's first examine some of the characterized repair

pathways and then consider how the cell integrates these systems into an overall

strategy for repair.

We can divide repair pathways into several categories.

Prevention of errors before they happen

Some enzymatic systems neutralize potentially damaging compounds before they

even react with DNA. One example of such a system is the detoxification of

superoxide radicals produced during oxidative damage to DNA: the enzyme

superoxide dismutasecatalyzes the conversion of the superoxide radicals into

hydrogen peroxide, and the enzyme catalase,in turn, converts the hydrogen peroxide

into water. Another error-prevention pathway depends on the protein product of the

mutTgene: this enzyme prevents the incorporation of 8-oxodG (see Figure 16-10),which arises by oxidation of dGTP, into DNA by hydrolyzing the triphosphate of

8-oxodG back to the monophosphate.

Direct reversal of damage

The most straightforward way to repair a lesion, once it occurs, is to reverse it directly,

thereby regenerating the normal base. Reversal is not always possible, because some

types of damage are essentially irreversible. In a few cases, however, lesions can be

repaired in this way. One case is a mutagenic photodimer caused by UV light (see

Figure 16-20). The cyclobutane pyrimidine photodimer can be repaired by aphotolyasethat has been found in bacteria and lower eukaryotes but not in humans.

The enzyme binds to the photodimer and splits it, in the presence of certain

wavelengths of visible light, to generate the original bases (Figure 16-25). This

enzyme cannot operate in the dark, so other repair pathways are required to remove

UV damage. A photolyase that reverses the 6-4 photoproducts has been detected in

plants andDrosophila.

Alkyltransferasesalso are enzymes taking part in the direct reversal of lesions. They

remove certain alkyl groups that have been added to the O-6 positions of guanine

(Figure 16-17) by such agents as NG and EMS. The methyltransferase from E. colihas been well studied. This enzyme transfers the methyl group from

O-6-methylguanine to a cysteine residue on the protein. When this happens, the

enzyme is inactivated, so this repair system can be saturated if the level of alkylation

is high enough.

Excision-repair pathways

General excision repair.

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Also termed nucleotide excision repair,this system includes the breaking of a

phosphodiester bond on either side of the lesion, on the same strand, resulting in the

excision of an oligonucleotide. This excision leaves a gap that is filled by repair

synthesis, and a ligase seals the breaks. In prokaryotes, 12 or 13 nucleotides are

removed; whereas, in eukaryotes, from 27 to 29 nucleotides are eliminated. Figure16-26depicts the incision pattern in each case.

InE. coli,the products of the uvrA, B,and Cgenes constitute the excinuclease. The

UvrA protein, which recognizes the damaged DNA, forms a complex with UvrB and

leads the UvrB subunit to the damage site before dissociating. The UvrC protein then

binds to UvrB. Each of these subunits makes an incision. The short DNA 12-mer is

unwound and released by another protein, helicase II. Figure 16-27shows a detailed

view of these excision events.

The human excinuclease is considerably more complex than its bacterial counterpart

and includes at least 17 proteins. However, the basic steps are the same as those inE.

coli.

Coupling of transcription and repair.

The involvement of TFIIH, a transcription factor, in excision repair underscores the

fact that transcription and repair are coupled. In both eukaryotes and prokaryotes,

there is a preferential repair of the transcribed strand of DNA for actively expressed

genes. Figure 16-28portrays a mechanism for this coupling.

Specific excision pathways.

Certain lesions are too subtle to cause a distortion large enough to be recognized by

the uvrABC-encoded general excision-repair system and its counterparts in higher

cells. Thus, additional excision pathways are necessary.

DNA glycosylase repair pathway (base-excision repair). DNA glycosylasesdo

not cleave phosphodiester bonds, but instead cleave N-glycosidic (basesugar) bonds,

liberating the altered base and generating an apurinic or an apyrimidinic site, both

called AP sites,because they are biochemically equivalent (see Figure 16-7). This

initial step is shown in Figure 16-29. The resulting Ap site is then repaired by an APendonuclease repair pathway (described in the next subsection).

Numerous DNA glycosylases exist. One, uracil-DNA glycosylase, removes uracil

from DNA. Uracil residues, which result from the spontaneous deamination of

cytosine (Figure 16-8), can lead to a C T transition if unrepaired. It is possible that

the natural pairing partner of adenine in DNA is thymine (5-methyluracil), rather than

uracil, to allow the recognition and excision of these uracil residues. If uracil were a

normal constituent of DNA, such repair would not be possible.

There is also a glycosylase that recognizes and excises hypoxanthine, the deamination

product of adenine. Other glycosylases remove alkylated bases (such as



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3-methyladenine, 3-methylguanine, and 7-methylguanine), ring-opened purines,

oxidatively damaged bases, and, in some organisms, UV photodimers. New

glycosylases are still being discovered.

AP endonuclease repair pathway. All cells have endonucleases that attack the

sites left after the spontaneous loss of single purine or pyrimidine residues. The AP

endonucleasesare vital to the cell, because, as noted earlier, spontaneous

depurination is a relatively frequent event. These enzymes introduce chain breaks by

cleaving the phosphodiester bonds at AP sites. This bond cleavage initiates an

excision-repair process mediated by three further enzymesan exonuclease, DNA

polymerase I, and DNA ligase (Figure 16-30).

Owing to the efficiency of the AP endonuclease repair pathway, it can be the final

step of other repair pathways. Thus, if damaged base pairs can be excised, leaving an

AP site, the AP endonucleases can complete the restoration to the wild type. This is

what happens in the DNA glycosylase repair pathway.

GO system. Two glycosylases, the products of the mutMand mutYgenes, work in

concert to prevent mutations arising from the 8-oxodG, or GO, lesion in DNA (see

Figure 16-10). Together with the product of the mutTgene mentioned earlier, these

glycosylases form the GO system. When GO lesions are generated in DNA by

spontaneous oxidative damage, a glycosylase encoded by mutMremoves the lesion

(Figure 16-31). Still, some GO lesions persist and mispair with adenine. A second

glycosylase, the product of the mutYgene, removes the adenine from this specific

mispair, leading to restoration of the correct cytosine by repair synthesis (mediated by

DNA polymerase I) and allowing subsequent removal of the GO lesion by the mutM

product.

The mutTproduct prevents incorporation of GO across from A. The human

counterparts of the mutT, mutY,and mutMgene products have been detected.

Postreplication repair

Mismatch repair.

Some repair pathways are capable of recognizing errors even after DNA replicationhas already occurred. One such system, termed the mismatch repair system,can

detect mismatches that occur in DNA replication. Suppose you were to design an

enzyme system that could repair replication errors. What would this system have to be

able to do? At least three things:

1.Recognize mismatched base pairs.

2.Determine which base in the mismatch is the incorrect one.

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3.Excise the incorrect base and carry out repair synthesis.

The second point is the crucial property of such a system. Unless it is capable of

discriminating between the correct and the incorrect bases, the mismatch repair

system could not determine which base to excise. If, for example, a GT mismatchoccurs as a replication error, how can the system determine whether G or T is

incorrect? Both are normal bases in DNA. But replication errors produce mismatches

on the newly synthesized strand, so it is the base on this strand that must be

recognized and excised.

To distinguish the old, template strand from the newly synthesized strand, the

mismatch repair system in bacteria takes advantage of the normal delay in the

postreplication methylation of the sequence

The methylating enzyme is adenine methylase,which creates 6-methyladenine on

each strand. However, it takes the adenine methylase several minutes to recognize and

modify the newly synthesized GATC stretches. During that interval, the mismatch

repair system can operate because it can now distinguish the old strand from the new

one by the methylation pattern. Methylating the 6-position of adenine does not affect

base pairing, and it provides a convenient tag that can be detected by other enzyme

systems. Figure 16-32shows the replication fork during mismatch correction. Notethat only the old strand is methylated at GATC sequences right after replication.

When the mismatched site has been identified, the mismatch repair system corrects

the error. Figure 16-33depicts a model of how the mismatch repair system carries out

the correction inE. coli.

The mismatch repair system has also been characterized in humans. Two of the

proteins, hMSH2 and hMLH1, are very similar to their bacterial counterparts, MutS

and MutL, respectively. Figure 16-34depicts how the hMSH2 protein, together with

the GT-binding protein (GTBP), binds to the mismatches and then recruits the othercomponents of the system, hPMS2 and hMLH1, to effect repair of the mismatch.

Recombinational repair.

The recAgene, which has a role in SOS bypass (Figure 16-19), also takes part in

postreplication repair. Here the DNA replication system stalls at a UV photodimer or

other blocking lesion and then restarts past the block, leaving a single-stranded gap. In

recombinational repair, this gap is patched by DNA cut from the sister molecule

(Figure 16-35a). This process seems to lead to few errors. SOS bypass, in contrast, is

highly mutagenic, as described earlier. Here the replication system continues past the

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lesion (Figures 16-19and 16-35b), accepting noncomplementary nucleotides for new

strand synthesis.

Strategy for repair

We can now assess the overall repair system strategy used by the cell. The manydifferent repair systems available to the cell are summarized in Table 16-2. It would

be convenient if enzymes could be used to directly reverse each specific lesion.

However, sometimes that is not chemically possible, and not every possible type of

DNA damage can be anticipated. Therefore, a general excision repair system is used

to remove any type of damaged base that causes a recognizable distortion in the

double helix. When lesions are too subtle to cause such a distortion, specific excision

systems, glycosylases, or removal systems are designed. To eliminate replication

errors, a postreplication mismatch repair system operates; finally, postreplication

recombinational systems eliminate gaps across from blocking lesions that have

escaped the other repair systems.

A number of repair pathways are induced in response to damage, such as the SOS

system (see Figure 16-19), and many of the proteins participating in the repair of

alkylation damage were discussed previously.

MESSAGE

Repair enzymes play a crucial role in reducing genetic damage in living cells. Thecell has many different repair pathways at its command to eliminate potentially

mutagenic errors.

Mutators

As the preceding description of repair processes indicates, normal cells are

programmed for error prevention. The repair processes are so efficient that the

observed basesubstitution rate is as low as 1010to 109per base pair per cell per

generation inE. coli.However, mutant strains with increased spontaneous mutation

rates have been detected. Such strains are termed mutators.In many cases, the

mutator phenotype is due to a defective repair system. In humans, these repair defects

often lead to serious diseases.

InE. coli,the mutator loci mutH, mutL, mutU,and mutSaffect components of the

postreplication mismatch repair system (see Figure 16-33), as does the damlocus,

which specifies the enzyme deoxyadenosine methylase. Strains that are Damcannot

methylate adenines at GATC sequences (see Figure 16-32), and so the mismatch

repair system can no longer discriminate between the template and the newly

synthesized strands. This failure to discriminate leads to a higher spontaneous

mutation rate.

Mutations in the mutYlocus result in GC

TA transversions, because many GAmispairs and all 8-oxodGA mispairs are unrepaired (see the GO system described

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earlier). The mutMgene encodes a glycosylase that removes 8-oxodG. Strains lacking

mutMare mutators for the GC TA transversion. Strains that are MutThave

elevated rates of the AT CG transversion, because they lack an activity that

prevents the incorporation of 8-oxodG across from adenine.

Strains that are Ungare missing the enzyme uracil DNA glycosylase. These mutants

cannot excise the uracil resulting from cytosine deaminations and, as a result, have

elevated levels of C T transitions. The mutDlocus is responsible for a very high

rate of mutagenesis (at least three orders of magnitude higher than normal). Mutations

at this locus affect the proofreading functions of DNA polymerase III (Chapter 8).

Figure 16-25. Repair of a UV-induced pyrimidine photodimer by a photoreactivating

enzyme, or photolyase. The enzyme recognizes the photodimer (here, a thymine

dimer) and binds to it. When light is present, the photolyase uses its energy to split the

dimer into the original monomers. (After J. D. Watson,Molecular Biology of the

Gene,3d ed. Copyright 1976 by W. A. Benjamin.)

UV-Induced Photodimers and Excision Repair.

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Figure 16-27. Schematic representation of events following incision by UvrABC

exinuclease inE. coli.First, the UvrA subunit recruits the UvrB subunit to the damage

site before dissociation. Then, as shown here, DNA helicase II mediates the release of

a segment of the DNA bounded by two nicks in the same strand of DNA. The UvrC

protein is also displaced at this point. The subsequent repair synthesis displaces UvrB.

(From E. C. Friedberg, G. C. Walker, and W. Seide,DNA Repair and Mutagenesis.

Copyright 1995 by the American Society for Microbiology.)

Figure 16-28.Nucleotide excision repair is coupled to transcription. This model for

coupled repair in mammalian cells shows RNA polymerase (pink) pausing when

encountering a lesion. It undergoes a conformational change, allowing the DNA

strands at the lesion site to reanneal. Protein factors aid in coupling by bringing TFIIH

and other factors to the site to carry out the incision, excision, and repair reactions.



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Then transcription can continue normally. (From P. C. Hanawalt, Science266, 2994,

2957.)

Figure 16-29. Action of DNA glycosylases. Glycosylases remove altered bases and

leave an AP site. The AP site is subsequently excised by the AP endonucleases

diagrammed in Figure 16-30. (After B. Lewin, Genes.Copyright 1983 by John

Wiley.)

Figure 16-30. Repair of AP (apurinic or apyrimidinic) sites. AP endonucleases

recognize AP sites and cut the phosphodiester bond. A stretch of DNA is removed by

an exonuclease, and the resulting gap is filled by DNA polymerase I and DNA ligase.

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