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Chapter 3 : Introduction to Mutagenesis
Category: Microbial Genetics and Molecular Biology
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One of the major reasons that so much effort has been devoted to analyzing DNA damage and understanding the biological mechanisms for its repair is that mutations can be introduced into DNA as a consequence of such damage. This chapter defines a number of terms that are used when discussing mutations and their biological consequences. It also describes examples of systems that have been developed to allow convenient analyses of mutations, describes use of site-specific adducts, and summarizes some of the simpler known mechanisms that can lead to the introduction of mutations in DNA. The process by which mutations are produced is referred to as mutagenesis. Mutagenesis that occurs without treatment of the organism with an exogenous mutagen is referred to as spontaneous mutagenesis. Spontaneous mutations can occur because of replication errors, or can arise as a consequence of lesions that are introduced into DNA during normal growth of the cell. To understand how chemically modified bases or other lesions give rise to mutations during DNA replication, it is instructive to consider how a normal DNA template is copied by a DNA polymerase. A fairly detailed understanding of the mechanisms of DNA synthesis has come from decades of biochemical studies of DNA polymerases and from analyses of crystal structures of these enzymes complexed to DNA and nucleotide substrates. These studies highlight the role of substrate shape in the selection of the correct deoxyribonucleotide for insertion opposite each template base.
Suppression of a nonsense mutation by a nonsense suppressor mutation.
Illustration of how a frameshift mutation results in a shift in the translational reading frame.
A portion of the T4 rII gene showing the number of mutations isolated at each site. Each square represents one occurrence at the indicated site. (Adapted from reference 25 .)
Base substitutions required to restore the glutamic acid codon at position 461 of β-galactosidase. Missense or nonsense mutations at coding position 461 result in the Lac- phenotype, but they can revert to the GAG codon by one of six base substitutions. In each case, one specific substitution restores the GAG codon ( 59 , 188 ). (Adapted from references 59 and 191 .)
The Ames test. A set of his auxotrophs of S. enterica serovar Typhimurium are mixed with the compound to be tested and plated on minimal-glucose plates containing a limiting amount of histidine. After 2 days of incubation, His+ revertants on each plate are counted. Mammalian metabolism is simulated by the addition of an extract of rat liver, termed the S9 supernatant. The S9 supernatant is prepared from rats that have been injected with a polychlorinated biphenyl mixture, Aroclor. (Adapted from reference 10 .)
Mutagenicity in the Ames test of aflatoxin B1. Strain TA1538 bears a different his mutation from that in strains TA100 and TA1535. Strain TA100 is a derivative of TA1535 that carries plasmid pKM101 (see chapter 15). (Adapted from reference 181 .)
Distribution of 3,738 mutations from G•C → A•T transitions in the lacI gene of E. coli. The number of independent occurrences at each site is indicated by the bar height. One amber and one ochre mutation were analyzed from each mutagenized culture, so that hot spots can be identified by comparing the frequency of mutation at an amber site with that at other amber sites and similarly by comparing the frequency of mutation at an ochre site with that at other ochre sites. See reference 56 for a discussion of amber mutation frequencies relative to ochre mutation frequencies. Large areas, instead of bar height, indicate that the number of occurrences is greater than 69, with the actual number shown in parentheses. Abbreviations: 4-NQO, 4-nitroquinoline-1-oxide; 2AP, 2-aminopurine; EMS, ethyl methanesulfonate; NG, N-methyl-N’-nitro-N-nitrosoguanidine. (Adapted from reference 56 .)
Secondary structure of a single-stranded DNA containing the supF tRNA gene sequence and showing the location of mutations that inactivate supF function. Sites of single (gold circles) and tandem (grey rectangles) base substitutions, insertions (gold triangles), and deletions (gold X) are indicated. (Adapted from reference 145 .)
Fusions of lacZ to lacI and the selection system used to detect deletions in lacI. (A) The deletion fuses the lacI gene to the lacZ gene. The resulting hybrid protein is missing the last 5 residues of the lac repressor and the first 23 residues of β-galactosidase but retains β-galactosidase activity ( 4 , 132 ). Q indicates the lacIq promoter. (B) Frameshift mutations 378 and S42, which are separated by 697 bp, have been crossed into the lacI-lacZ fusion. Only deletions can restore the Lac+ character. The principal deletions that were detected were of the a or b type. (Adapted from reference 3 .)
Schematic representation of a shuttle vector genetic map. Mammalian ori represents DNA sequences from animal virus replication origins allowing replication in mammalian cells by using a viral trans-acting protein. The selection gene codes for a protein allowing selection and maintenance of this vector in mammalian cells. The bacterial ori represents DNA sequences from a bacterial plasmid or bacteriophage. The antibiotic resistance gene codes for a protein allowing the selection and maintenance of the vector in bacteria. The target gene represents the DNA sequences used for detecting mutants (see also Table 3–5 ). (Adapted from reference 231 .)
General scheme of the various protocols involving shuttle vectors. 1, Vector DNA is first transfected into the mammalian host cell. 2, By using an SV40-based plasmid, transient replication occurs in 2 to 4 days after transfection. 3, By using an Epstein-Barr virus-based plasmid and selecting for antibiotic resistance, established cell lines replicating the vector as an epi-some are isolated. 4, By using a nonreplicating vector and selecting for a gene on the vector, cell lines containing integrated shuttle vector sequences are isolated. 5, Replicated and mutated vector sequences are recovered as low-molecular-weight (L.M.W.) DNA, which is then transfected into host bacteria cells. DNA alterations in the vector target gene are analyzed from isolated colonies or plaques. (Adapted from reference 231 .)
Strategies involving PCR to analyze mutations in a target gene in a mammalian cell line. These involve first selecting for mutants (e.g., 6-thioguanine resistance) and then either making cDNA and amplifying the sequence or directly amplifying fragments of the genomic DNA and sequencing.
Analyses of mutagenesis in transgenic mice. See the text for details.
Principle of Spi— selection. Growth of wild-type X phage is restricted in E. coli cells carrying P2 phage DNA in the chromosome, i.e., P2 lysogen. This phenomenon is called P2 interference. Mutant X phages deficient in both red and gam gene functions grow well in P2 lysogen and display the Spi— phenotype as long as they carry a χ site and the host strain is recA+. Since simultaneous inactivation of two genes is often induced by deletions in the region, Spi— selection preferentially detects deletion mutants of λ DNA ( 121 ). (Adapted from reference 205 .)
Potential mechanisms of loss of heterozygosity (LOH). Various mutagenic events can lead to the loss of function of a second allele of an autosomal gene. LOH can result from locus-restricted events such as gene conversion or multilocus events such as large deletion, mitotic recombination, or mitotic nondisjunction with or without duplication of the remaining chromosome. In the upper picture, two homologous chromosomes that contain a heterozygosity for gene A are shown. (Adapted from reference 274 .)
(A) The generation of MassEXTEND reaction products as part of the MassARRAY system. Prior to the MassEXTEND reaction, genomic DNA containing the SNP site of interest is amplified by PCR and shrimp alkaline phosphatase is added to samples to dephosphorylate any residual amplification nucleotides and to prevent their future incorporation and interference with the primer extension assay (not shown). The MassEXTEND primer, DNA polymerase, and a cocktail mixture of dNTPs and ddNTPs are added to initiate the primer extension reaction. This reaction generates allele-specific primer extension products that are generally 1 to 4 bases longer than the original MassEXTEND primer. A common MassEXTEND primer that identifies both alleles is hybridized directly or closely adjacent to the polymorphic site. Nucleotide mixtures are selected to maximize mass differences for all potential MassEXTEND products. Appropriate deoxynucleotides are incorporated through the polymorphic site until a single dideoxynucleotide is incorporated and the reaction terminates. Since the termination point and number of nucleotides is sequence specific, the mass of the extension products generated for allele 1 and allele 2 can be used to identify the possible variants by using MALDI-TOF analysis. (B) Spectral analysis of MassEXTEND reaction primer extension products. Each addition of a nucleotide to the primer extension product increases the mass by 289 to 329 Da, depending on the nucleotide added. The mass difference is easily resolved by MALDI-TOF, which has the ability to detect differences as small as 3 Da. Thus, alleles differing by a single nucleotide are readily discriminated. (Adapted from Application Notes, Bulletin 1021, Sequenom, Inc., with permission.)
Methods for the construction of site-specifically modified genomes. The procedure shown on the left uses DNA ligase to insert an adducted oligonucleotide into a complementary site in a gapped heteroduplex genome. The product can be used directly to study the genetic effect of the lesion in double-stranded DNA, or if the strand opposing the adduct contains a nonligatable nick, the genome can be denatured and the biological effects of an adduct situated in a single-stranded DNA can be determined. One modification of the approach depicted on the right has been to use DNA polymerase to join an adducted dNTP onto the 3’ end of an unmodified oligonucleotide previously annealed to a single-stranded genome. Synthesis of the site-specifically modified duplex vector is completed upon subsequent addition of unmodified dNTPs. (Adapted from reference 88 .)
The base-pairing scheme dictating the double-helical structure of DNA can accommodate mispairs like the G•T Hoogsteen base pair shown here. Hoogsteen pairs have a different shape from the normal Watson-Crick pairs, and those arising from mistakes during replication are typically subject to proofreading. An exception is the oxidative lesion 8-oxoguanosine, which forms a stable Hoogsteen base pair with A that is not subject to proofreading ( 39 , 119 ) and is consequently highly mutagenic (see chapter 4).
Geometric characteristics of Watson-Crick and mismatched base pairs. The figure is based on X-ray crystallography of duplex B-DNA oligonucleotides. The striking geometric identity of the Watson-Crick base pairs (A and B) is not matched by the A•C (C) and G•T (D) wobble pairs or by the G(anti)-A(syn) pair (E). The G-T pairing shown in panel D is a Hoogsteen base pair. (Adapted from references 84 and 131 .)
The crystal structure of the Klenow fragment of E. coli DNA polymerase I revealed a protein fold shaped like a right hand, with fingers, thumb, and palm subdomains ( 211 ). The polymerase active site is located at the junction of the palm and fingers, and the proofreading 3’ to 5’ exonuclease is located in a separate region at the N terminus of the protein (cf. Fig. 3–29 ). Many different DNA polymerases, and the monomeric RNA polymerases from bacteriophages, have a similar shape resembling a right hand ( 248 ).
DNA Pol β is a repair polymerase that functions in the gap-filling reaction during base excision repair. The crystal structure of Pol β inserting a nucleotide into DNA containing a single-nucleotide gap shows that the polymerase contacts the DNA ends on both sides of the gap ( 233 ). In complex with DNA and nucleotide substrates, Pol β adopts a closed conformation that aligns the nucleotide for insertion at the 3’ end of the primer DNA strand ( 20 ).
DNA polymerases catalyze the addition of nucleoside monophosphates to the 3’ end of a primer DNA strand by a nucleophilic displacement mechanism featuring two metal ions ( 248 ). The 3’ OH of the primer strand is activated for nucleophilic attack of the α-phosphorus of an NTP, resulting in the release of pyrophosphate and incorporation of a nucleoside monophosphate into the growing DNA strand. The metals serve to align the reacting molecules, activate the 3’ OH, and counteract the growing charge on the pyrophosphate leaving group ( 69 ).
DNA polymerases utilize an induced-fit mechanism for selecting the correct nucleotide for incorporation into DNA ( 20 , 70 , 247 ). The closure of the fingers subdomain when nucleotides bind in the polymerase active site provides a means of sensing the shape of the nascent base pair, which is an important means of substrate selection ( 142 ).
In the closed conformation, the active site of T7 DNA polymerase forms a narrow slot for the incoming nucleotide and a template base. The shape and surface chemistry of the closed polymerase are compatible with Watson-Crick base pairs, whereas mispairs fit poorly in the active site and prevent the fingers from closing ( 69 ). Substrate-induced conformational changes have been observed in crystal structures of a variety of DNA and RNA polymerases ( 20 , 248 ).
The phosphoryl transfer reaction catalyzed by DNA and RNA polymerases features charged intermediates that are stabilized by basic amino acids and divalent metals bound in the polymerase active site to catalyze DNA synthesis. The actual structure of the transition state for this reaction is likely to be intermediate between two extremes, a dissociative metaphosphate-like state (A) and an associative pentacoordinate state (B) ( 1 ). The induced-fit-type mechanism for the catalytic selectivity of DNA polymerases, shown in Fig. 3–23 and 3-24 , suggests how the structure of the polymerase is adapted to the structure of the transition state for nucleotide incorporation into DNA.
The O-helix within the fingers of Pol A family DNA polymerases contains conserved residues that interact with the incoming nucleotide and template base and strongly influence the rate and fidelity of DNA synthesis (see Fig. 3–25 ).
The nucleobase isosteres 2,4-difluorotoluene (compound F) and 4-methylbenzimidazole (compound Z) mimic the shape of the natural DNA bases T and A, respectively, but lack hydrogen-bonding capability. Nonetheless, nonpolar nucleoside mimics containing these unnatural bases are selectively incorporated by the Klenow fragment DNA polymerase ( 142 ). This result shows that the shape of the nascent base pair is an important determinant of the fidelity of DNA synthesis, even in the absence of Watson-Crick hydrogen bonding between the template base and the incoming nucleotide.
Exonucleolytic proofreading is a mechanism for removing misincorporated nucleotides during DNA synthesis, increasing the accuracy of copying a DNA template approximately 100-fold. (A) The 3’ to 5’ exonuclease of the Klenow fragment DNA polymerase is located far from the polymerase active site, necessitating a large shift in the bound DNA during the transition from DNA synthesis (left) and proofreading (right) ( 22 ). DNA synthesis is slowed by the misincorporation of a nucleotide because the 3’-OH is misaligned for the subsequent round of nucleotide incorporation into DNA. This slowing of DNA synthesis favors the dissociation of the DNA from the polymerase site and rebinding in the 3’ to 5’ proofreading exonuclease site. After one or more nucleotides are removed from the 3’ end of the primer strand, DNA synthesis resumes in the polymerase active site. (B) A series of minor-groove interactions with the base pairs located in the polymerase active site may provide the mechanism for detecting mismatched base pairs, triggering the movement of DNA to the exonuclease and the proofreading reaction. A wobble or Hoogsteen base pair resulting from misinsertion of a nucleotide by the polymerase would not accommodate these interactions with the minor groove.
The error-prone DNA polymerases of the polymerase Y superfamily contain five conserved sequence motifs that are different from those of other DNA polymerases. The discovery of the Y polymerase superfamily resulted from the isolation of several novel DNA polymerases with the unusual ability to synthesize DNA past chemically modified nucleotides in a DNA template. It was subsequently realized that polymerases with these conserved motifs are widespread in all kingdoms of life ( 208 ). (Adapted from reference 126 .)
The error-prone Y family DNA polymerases have a shape resembling a right hand, with fingers, thumb, and palm subdomains, but the active site of these enzymes is less constraining than the closed active site of high-fidelity DNA polymerases ( Fig. 3–24 and 3-25 ). The crystal structure of the S. sulfataricus Dpo4 polymerase complexed to DNA showed that two nucleotides on the template strand could fit into the polymerase active (note the unpaired 5’ nucleotide bound near the fingers), providing an explanation for the bypass of CPD and other bulky lesions ( 168 ). A C-terminal domain of Y family polymerases, alternatively called the little finger or polymerase-associated domain, binds to DNA and significantly influences DNA lesion bypass activity ( 32 ).
(A) UV exposure damages DNA, causing a particularly insidious lesion, the CPD, at dipyrimidine sequences in DNA. The covalent linkage of two adjacent bases in a CPD impairs DNA synthesis by replicative polymerases ( 163 ). (B) Specialized polymerases of the Y superfamily can bypass CPD lesions by virtue of their more accommodating active sites ( Fig. 3–31 ). The crystal structure of the S. sulfataricus Dpo4 polymerase in complex with DNA containing a CPD lesion shows how the enzyme can catalyze the templated insertion of A across from the 3’ T of CPD, by accommodating both thymines of the CPD at once in the polymerase active site ( 167 ).
Mutational intermediates for substitution and frameshift errors that involve primer-template misalignments. (Adapted from reference 147 .)
Misalignment between tandem repeats improved by a palindrome. In each case the deletion is produced by misaligning arrow 1’ with arrow 2. Arrow 1 is a repeat of arrow 2; arrow 1 ‘ is the normal complement of arrow 1. (A) The palindrome brings arrow 2 to precisely the misaligned position that produces a deletion ( 122 ). (B) The palindrome brings arrow 2 adjacent to the misaligned position ( 143 ). (C) The palindrome brings arrow 2 closer ( 227 ). (Adapted from reference 227 .)
Model for the production of templated T4 rIIB mutations. The complex mutations termed mutant 1 and mutant 2 are rationalized as being due to the formation of a palindromic intermediate between two quasi-homologous sequences. Changed bases are in gold, and Δ indicates a deleted base. (Adapted from references 63 and 77 .)