Chapter Review

Key Terms

Terms in bold are defined in the glossary.

Problems

Data Analysis Problem

  • 22. Mutagenesis in Escherichia coli Many mutagenic compounds act by alkylating the bases in DNA. The alkylating agent R7000 (7-methoxy-2-nitronaphtho[2,1-b]furan) is an extremely potent mutagen.

    A molecule labeled R 7000 has a central benzene ring that shares its right side with the left side of a five-membered ring with O substituted for C at its lower right vertex, a double bond at its upper right side, and a right side vertex bonded to N O 2. The central benzene ring shares its upper left side with the lower right side of a left-hand benzene ring that has an upper left vertex bonded to O further bonded to C H 3.

    In vivo, R7000 is activated by the enzyme nitroreductase, and this more reactive form covalently attaches to DNA — primarily, but not exclusively, to GCupper G identical-to upper C base pairs.

    In a 1996 study, Quillardet, Touati, and Hofnung explored the mechanisms by which R7000 causes mutations in E. coli. They compared the genotoxic activity of R7000 in two strains of E. coli: the wild-type (Uvr+)left-parenthesis Uvr Superscript plus Baseline right-parenthesis and mutants lacking uvrA activity (Uvr)left-parenthesis Uvr Superscript minus Baseline right-parenthesis. They first measured rates of mutagenesis. Rifampicin is an inhibitor of RNA polymerase. In its presence, cells will not grow unless certain mutations occur in the gene encoding RNA polymerase; the appearance of rifampicin-resistant colonies thus provides a useful measure of mutagenesis rates.

    The investigators determined the effects of different concentrations of R7000. Their results are shown in the following graph:

    A graph plots rifampicin-resistant mutants produced against R 7000 (micrograms / m L).
    1. Why are some mutants produced even when no R7000 is present?

      Quillardet and colleagues also measured the survival rate of bacteria treated with different concentrations of R7000, with the following results:

      A graph plots survival (percent) against R 7000 (micrograms / m L).
    2. Explain how treatment with R7000 is lethal to cells.
    3. Explain the differences in the mutagenesis curves and in the survival curves for the two types of bacteria, Uvr+Uvr Superscript plus and UvrUvr Superscript minus, as shown in the graphs.

      The researchers went on to measure the amount of R7000 covalently attached to the DNA in Uvr+Uvr Superscript plus and UvrUvr Superscript minus E. coli. They incubated bacteria with [3H]R7000left-bracket Superscript 3 Baseline upper H right-bracket upper R 7000 for 10 or 70 minutes, extracted the DNA, and measured its 3Hcubed upper H content in counts per minute (cpm) per microgram of DNA.

      3Hcubed bold upper H in DNA (cpm/μg)bold left-parenthesis bold cpm bold slash bold-italic mu bold g bold right-parenthesis

      Time (min)

      Uvr+bold Uv bold r Superscript bold plus

      Uvrbold Uv bold r Superscript bold minus

      10

      76

      159

      70

      69

      228

    4. Explain why the amount of 3Hcubed upper H drops over time in the Uvr+Uvr Superscript plus strain and rises over time in the UvrUvr Superscript minus strain.

      Quillardet and colleagues then examined the particular DNA sequence changes caused by R7000 in the Uvr+Uvr Superscript plus and UvrUvr Superscript minus bacteria. For this, they used six different strains of E. coli, each with a different point mutation in the lacZ gene, which encodes β-galactosidase. Cells with any of these mutations have a nonfunctional β-galactosidase and are unable to metabolize lactose (i.e., a Lac phenotypeLac Superscript minus Baseline phenotype). Each type of point mutation required a specific reverse mutation to restore lacZ gene function and Lac+ phenotypeLac Superscript plus Baseline phenotype. By plating cells on a medium containing lactose as the sole carbon source, the researchers selected for these reverse-mutated, Lac+ cellsLac Superscript plus Baseline cells. And by counting the number of Lac+ cellsLac Superscript plus Baseline cells following mutagenesis of a particular strain, they could measure the frequency of each type of mutation.

      First, they looked at the mutation spectrum in Uvr cellsUvr Superscript minus Baseline cells. The following table shows the results for the six strains, CC101 through CC106 (with the point mutation required to produce Lac+ cellsLac Superscript plus Baseline cells indicated in parentheses).

    Number of Lac+ cells (average±SD)bold Number of bold bold La bold c Superscript bold plus Baseline bold bold cells bold bold left-parenthesis bold average bold plus-or-minus bold SD bold right-parenthesis
    R7000 (μg/mL)bold upper R bold 7000 bold left-parenthesis bold-italic mu bold g slash bold mL right-parenthesis CC101 (ATbold upper A bold box drawings double horizontal bold upper T to CGbold upper C bold identical-to bold upper G) CC102 (GCbold upper G bold identical-to bold upper C to ATbold upper A bold box drawings double horizontal bold upper T) CC103 (GCbold upper G bold identical-to bold upper C to CGbold upper C bold identical-to bold upper G) CC104 (GCbold upper G bold identical-to bold upper C to TAbold upper T bold box drawings double horizontal bold upper A) CC105 (ATbold upper A bold box drawings double horizontal bold upper T to TAbold upper T bold box drawings double horizontal bold upper A) CC106 (ATbold upper A bold box drawings double horizontal bold upper T to GCbold upper G bold identical-to bold upper C)

    0

    6±36 plus-or-minus 3

    11±911 plus-or-minus 9

    2±12 plus-or-minus 1

    5±35 plus-or-minus 3

    2±12 plus-or-minus 1

    1±11 plus-or-minus 1

    0.075

    24±1924 plus-or-minus 19

    34±334 plus-or-minus 3

    8±48 plus-or-minus 4

    82±2382 plus-or-minus 23

    40±1440 plus-or-minus 14

    4±24 plus-or-minus 2

    0.15

    24±424 plus-or-minus 4

    26±226 plus-or-minus 2

    9±59 plus-or-minus 5

    180±71180 plus-or-minus 71

    130±50130 plus-or-minus 50

    3±23 plus-or-minus 2

    1. Which types of mutation show significant increases above the background rate due to treatment with R7000? Provide a plausible explanation for why some have higher frequencies than others.
    2. Can all of the mutations you listed in (e) be explained as resulting from covalent attachment of R7000 to a GCupper G identical-to upper C base pair? Explain your reasoning.
    3. Figure 25-26b shows how methylation of guanine residues can lead to a GCupper G identical-to upper C to ATupper A box drawings double horizontal upper T mutation. Using a similar pathway, show how an R7000–G adduct could lead to the GCupper G identical-to upper C to ATupper A box drawings double horizontal upper T or TAupper T box drawings double horizontal upper A mutations shown above. Which base pairs with the R7000–G adduct?

      The results for the Uvr+Uvr Superscript plus bacteria are shown in the following table.

    Number of Lac+ cells (average±SD)bold Number of bold bold La bold c Superscript bold plus Baseline bold bold cells bold bold left-parenthesis bold average bold plus-or-minus bold SD bold right-parenthesis
    R7000bold upper R bold 7000 (μg/mLbold-italic mu bold g bold slash bold mL) CC101 (ATbold upper A bold box drawings double horizontal bold upper T to CGbold upper C bold identical-to bold upper G) CC102 (GCbold upper G bold identical-to bold upper C to ATbold upper A bold box drawings double horizontal bold upper T) CC103 (GCbold upper G bold identical-to bold upper C to CGbold upper C bold identical-to bold upper G) CC104 (GCbold upper G bold identical-to bold upper C to TAbold upper T bold box drawings double horizontal bold upper A) CC105 (ATbold upper A bold box drawings double horizontal bold upper T to TAbold upper T bold box drawings double horizontal bold upper A) CC106 (ATbold upper A bold box drawings double horizontal bold upper T to GCbold upper G bold identical-to bold upper C)

    0

    2±22 plus-or-minus 2

    10±910 plus-or-minus 9

    3±33 plus-or-minus 3

    4±24 plus-or-minus 2

    6±16 plus-or-minus 1

    0.5±10.5 plus-or-minus 1

    1

    7±67 plus-or-minus 6

    21±921 plus-or-minus 9

    8±38 plus-or-minus 3

    23±1523 plus-or-minus 15

    13±113 plus-or-minus 1

    1±11 plus-or-minus 1

    5

    4±34 plus-or-minus 3

    15±715 plus-or-minus 7

    22±222 plus-or-minus 2

    68±2568 plus-or-minus 25

    67±1467 plus-or-minus 14

    1±11 plus-or-minus 1

    1. Do these results show that all mutation types are repaired with equal fidelity? Provide a plausible explanation for your answer.

References

  • Quillardet, P., E. Touati, and M. Hofnung. 1996. Influence of the uvr-dependent nucleotide-excision repair on DNA adducts formation and mutagenic spectrum of a potent genotoxic agent: 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000). Mutat. Res. 358:113–122.