Vol. 137, No. 1
JouRNAL o0 BACTEOLOGY, Jan. 1979, p. 234-242 0021-9193/78/01-0234/09$02.00/0
Pyrimidine Dimer Excision in Escherichia coli Strains Deficient in Exonucleases V and VII and in the 5'-. 3' Exonuclease of DNA Polymerase I JOHN W. CHASE,`* WARREN E. MASKER,2 AND JANET B. MURPHY' Department of Molecular Biology, Division of Biological Science8, Albert Einstein College ofMedicine, Bronx, New York 10461,' and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 378302
Received for publication 25 July 1978
An isogenic series of Escherichia coli strains deficient in various combinations of three 5' -. 3' exonucleases (exonuclease V, exonuclease VII, and the 5' -. 3' exonuclease of DNA polymerase I) was constructed and examined for the ability to excise pyrimidine dimers after UV irradiation. Although the recB and recC mutations (deficient in exonuclease V) proved to be incompatible with the poLA(Ex) mutation (deficient in the 5' -. 3' exonuclease of DNA polymerase I), it was possible to reduce the level of the recB,C exonuclease by the use of temperature-sensitive recB270 recC271 mutants. It was found that, by employing strains deficient in exonuclease V, postirradiation DNA degradation could be reduced and dimer excision measurements could be facilitated. Mutants deficient in exonuclease V were found to excise dimers at a rate comparable to that of the wild type. Mutants deficient in exonuclease V and the 5' -* 3' exonuclease of DNA polymerase I are slightly slower than the wild type at removing dimers accumulated after doses in excess of 40 J/m2. However, although strains with reduced levels of exonuclease VII excised dimers at the same rate as the wild type, the addition of an exonuclease VII deficiency to a strain with reduced levels of exonuclease V and the 5' -. 3' exonuclease of DNA polymerase I caused a marked decrease in the rate and extent of dimer excision. These observations support previous indications that the 5' -. 3' exonuclease of DNA polymerase I is important in dimer removal and also suggest a role for exonuclease VII in the excision repair process. Previous studies (4, 8) have shown that Escherichia coli strains containing the polA(Ex) mutation (deficient in the 5' -a 3' exonuclease of DNA polymerase I) are somewhat deficient in the excision of pyrimidine dimers from UV-irradiated DNA, a process important in the dark repair of DNA damage (12, 13). It is also known that these strains suffer extensive DNA degradation after UV irradiation with doses in excess of 40 J/m2. Since there is no assurance that DNA containing pyrimidine dimers is degraded at the same rate as DNA without lesions, significant postirradiation DNA degradation can cause potentially serious complications in the interpretation of dimer excision measurements. To circumvent this difficulty and to explore the possibility that exonuclease V (11, 26) may play a role in dimer excision, we constructed a series of strains deficient in the recB,C enzyme (exonuclease V), as well as in two other 5' -- 3' exonucleases (the 5' -. 3' exonuclease of DNA polymerase I and exonuclease VII) known to be 234
capable of dimer excision in vitro (5, 14). Although the polA(Ex) mutation was found to be incompatible with recB21 mutants, it was possible to construct conditionally lethal recB270 recC271 mutants (22) with additional mutation in polA(Ex). These strains showed significantly reduced levels of postirradiation DNA degradation and allowed an investigation of the effect of 5' -. 3' exonuclease deficiencies on the rate and extent of dimer excision over a wide UV dose range (0 to 150 J/m2) without the complications of excessive DNA degradation. A comparison of the kinetics of dimer excision between the wild type and recB270 recC271 mutants after irradiation with high doses (90 J/m2) confirmed previous conclusions (21) suggesting no significant excision deficiency associated with reduced levels of exonuclease V. Mutants deficient in both exonuclease V and the 5' -. 3' exonuclease of DNA polymerase I excised dimers as rapidly as wild-type cells after a low dose of UV (20 J/m2) but were found to be
I9 *'L. 137, 1979
DNA REPAIR IN EXONUCLEASE-DEFICIENT MUTANTS
slower than the wild type at repairing damage introduced by higher levels of irradiation. Mutants deficient in exonucleases V and VII excised dimers as efficiently as wild-type cells at all UV doses tested. However, the addition of an exonuclease VII deficiency to a recB270 recC271 poUA(Ex) mutant caused greatly reduced viability at the restrictive temperature and a marked decrease in the rate at which dimers were removed after exposure to UV doses greater than 40 J/m2. Although this strain is highly sensitive to even low doses of UV, it is identical to the wild type in its ability to cope with pyrimidine diimers accumulated after exposure to 20 J of UV per m2. These results confirm previous indications (4, 8, 10) that the 5' -. 3' exonuclease of DNA polymerase I is important in dimer removal. Moreover, these data indicate a role for exonuclease VII in the excision repair process and suggest that this enzyme may substitute for the 5'-* 3' exonuclease of DNA polymerase I or may operate in an alternate excision repair pathway.
MATERIALS AND METHODS Bacterial and phage strains. The strains of E. coli K-12 used are listed in Table 1. Phage PlCMcIrlO0 was used for generalized transductions (19). Strains KLC277 and KLC290 were constructed from strains KLC147 and KLC148, respectively, by transduction with PlCMcIrlO0 grown on strain SK129 and selection for Thy' recombinants. These recombinants were subsequently tested for sensitivity to UV irradiation and mitomycin C. To confirm the identification, strains were assayed directly for exonucleases V and VII. Fractions I and V prepared by the procedure of Eichler and Lehman (9) were assayed for exonuclease V activity. Exonuclease V activity was not detected (
JouRNAL o0 BACTEOLOGY, Jan. 1979, p. 234-242 0021-9193/78/01-0234/09$02.00/0
Pyrimidine Dimer Excision in Escherichia coli Strains Deficient in Exonucleases V and VII and in the 5'-. 3' Exonuclease of DNA Polymerase I JOHN W. CHASE,`* WARREN E. MASKER,2 AND JANET B. MURPHY' Department of Molecular Biology, Division of Biological Science8, Albert Einstein College ofMedicine, Bronx, New York 10461,' and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 378302
Received for publication 25 July 1978
An isogenic series of Escherichia coli strains deficient in various combinations of three 5' -. 3' exonucleases (exonuclease V, exonuclease VII, and the 5' -. 3' exonuclease of DNA polymerase I) was constructed and examined for the ability to excise pyrimidine dimers after UV irradiation. Although the recB and recC mutations (deficient in exonuclease V) proved to be incompatible with the poLA(Ex) mutation (deficient in the 5' -. 3' exonuclease of DNA polymerase I), it was possible to reduce the level of the recB,C exonuclease by the use of temperature-sensitive recB270 recC271 mutants. It was found that, by employing strains deficient in exonuclease V, postirradiation DNA degradation could be reduced and dimer excision measurements could be facilitated. Mutants deficient in exonuclease V were found to excise dimers at a rate comparable to that of the wild type. Mutants deficient in exonuclease V and the 5' -* 3' exonuclease of DNA polymerase I are slightly slower than the wild type at removing dimers accumulated after doses in excess of 40 J/m2. However, although strains with reduced levels of exonuclease VII excised dimers at the same rate as the wild type, the addition of an exonuclease VII deficiency to a strain with reduced levels of exonuclease V and the 5' -. 3' exonuclease of DNA polymerase I caused a marked decrease in the rate and extent of dimer excision. These observations support previous indications that the 5' -. 3' exonuclease of DNA polymerase I is important in dimer removal and also suggest a role for exonuclease VII in the excision repair process. Previous studies (4, 8) have shown that Escherichia coli strains containing the polA(Ex) mutation (deficient in the 5' -a 3' exonuclease of DNA polymerase I) are somewhat deficient in the excision of pyrimidine dimers from UV-irradiated DNA, a process important in the dark repair of DNA damage (12, 13). It is also known that these strains suffer extensive DNA degradation after UV irradiation with doses in excess of 40 J/m2. Since there is no assurance that DNA containing pyrimidine dimers is degraded at the same rate as DNA without lesions, significant postirradiation DNA degradation can cause potentially serious complications in the interpretation of dimer excision measurements. To circumvent this difficulty and to explore the possibility that exonuclease V (11, 26) may play a role in dimer excision, we constructed a series of strains deficient in the recB,C enzyme (exonuclease V), as well as in two other 5' -- 3' exonucleases (the 5' -. 3' exonuclease of DNA polymerase I and exonuclease VII) known to be 234
capable of dimer excision in vitro (5, 14). Although the polA(Ex) mutation was found to be incompatible with recB21 mutants, it was possible to construct conditionally lethal recB270 recC271 mutants (22) with additional mutation in polA(Ex). These strains showed significantly reduced levels of postirradiation DNA degradation and allowed an investigation of the effect of 5' -. 3' exonuclease deficiencies on the rate and extent of dimer excision over a wide UV dose range (0 to 150 J/m2) without the complications of excessive DNA degradation. A comparison of the kinetics of dimer excision between the wild type and recB270 recC271 mutants after irradiation with high doses (90 J/m2) confirmed previous conclusions (21) suggesting no significant excision deficiency associated with reduced levels of exonuclease V. Mutants deficient in both exonuclease V and the 5' -. 3' exonuclease of DNA polymerase I excised dimers as rapidly as wild-type cells after a low dose of UV (20 J/m2) but were found to be
I9 *'L. 137, 1979
DNA REPAIR IN EXONUCLEASE-DEFICIENT MUTANTS
slower than the wild type at repairing damage introduced by higher levels of irradiation. Mutants deficient in exonucleases V and VII excised dimers as efficiently as wild-type cells at all UV doses tested. However, the addition of an exonuclease VII deficiency to a recB270 recC271 poUA(Ex) mutant caused greatly reduced viability at the restrictive temperature and a marked decrease in the rate at which dimers were removed after exposure to UV doses greater than 40 J/m2. Although this strain is highly sensitive to even low doses of UV, it is identical to the wild type in its ability to cope with pyrimidine diimers accumulated after exposure to 20 J of UV per m2. These results confirm previous indications (4, 8, 10) that the 5' -. 3' exonuclease of DNA polymerase I is important in dimer removal. Moreover, these data indicate a role for exonuclease VII in the excision repair process and suggest that this enzyme may substitute for the 5'-* 3' exonuclease of DNA polymerase I or may operate in an alternate excision repair pathway.
MATERIALS AND METHODS Bacterial and phage strains. The strains of E. coli K-12 used are listed in Table 1. Phage PlCMcIrlO0 was used for generalized transductions (19). Strains KLC277 and KLC290 were constructed from strains KLC147 and KLC148, respectively, by transduction with PlCMcIrlO0 grown on strain SK129 and selection for Thy' recombinants. These recombinants were subsequently tested for sensitivity to UV irradiation and mitomycin C. To confirm the identification, strains were assayed directly for exonucleases V and VII. Fractions I and V prepared by the procedure of Eichler and Lehman (9) were assayed for exonuclease V activity. Exonuclease V activity was not detected (
