Positive Charges Are Important for the SOS Constitutive Phenotype in and Mutants of Escherichia Coli K-12

Overview

Journal J Bacteriol

Publisher American Society for Microbiology

Specialty Microbiology

Date 2022 Apr 20

PMID 35442066

Authors

Steven Van Alstine

Steven J Sandler

Affiliations

Soon will be listed here.

Abstract

In Escherichia coli K-12, RecA binds to single-strand DNA (ssDNA) created by DNA damage to form a protein-DNA helical filament that serves to catalyze LexA autoproteolysis, which induces the SOS response. The SOS constitutive (SOS) mutations (E38K) and (Q184K) are both on the outside of the RecA filament, opposite to the face that binds DNA. (E38K) is also able to suppress the UV sensitivity caused by mutations. Both SOS expression and suppression are thought to be due to RecA730's ability to compete better for ssDNA coated with ssDNA-binding protein than the wild type. We tested whether other positively charged residues at these two positions would lead to SOS expression and suppression. We found that 5/6 positively charged residues were SOS and 4/5 of these were also suppressors. While other mutations at these two positions (and others) were suppressors, none were SOS. Three suppressors could be made moderately SOS by adding a operator mutation. We hypothesize two mechanisms for SOS expression: the first suggests that the positive charge at positions 38 and 184 attract negatively charged molecules that block interactions that would destabilize the RecA-DNA filament, and the second involves more stable filaments caused by increases in mutant RecA concentration. In Escherichia coli K-12, SOS constitutive (SOS) mutants of turn on the SOS response in the absence of DNA damage. Some SOS mutants are also able to indirectly suppress the UV sensitivity of mutations. Two SOS mutations, (E38K) and (Q184K), define a surface on the RecA-DNA filament opposite the surface that binds DNA. Both introduce positive charges, and is a suppressor. We tested whether the positive charge at these two positions was required for SOS expression and suppression. We found a high correlation between the positive charge, SOS expression and suppression. We also found several other mutations (different types) that provide suppression but no SOS expression.

References

Volkert M, Margossian L, Clark A . Evidence that rnmB is the operator of the Escherichia coli recA gene. Proc Natl Acad Sci U S A. 1981; 78(3):1786-90. PMC: 319219. DOI: 10.1073/pnas.78.3.1786. View

TESSMAN E, Peterson P . Isolation of protease-proficient, recombinase-deficient recA mutants of Escherichia coli K-12. J Bacteriol. 1985; 163(2):688-95. PMC: 219176. DOI: 10.1128/jb.163.2.688-695.1985. View

Cox M . Regulation of bacterial RecA protein function. Crit Rev Biochem Mol Biol. 2007; 42(1):41-63. DOI: 10.1080/10409230701260258. View

Long J, Renzette N, Sandler S . Suppression of constitutive SOS expression by recA4162 (I298V) and recA4164 (L126V) requires UvrD and RecX in Escherichia coli K-12. Mol Microbiol. 2009; 73(2):226-39. PMC: 2758083. DOI: 10.1111/j.1365-2958.2009.06765.x. View

Courcelle J, Khodursky A, Peter B, Brown P, HANAWALT P . Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli. Genetics. 2001; 158(1):41-64. PMC: 1461638. DOI: 10.1093/genetics/158.1.41. View

Menetski J, Kowalczykowski S . Interaction of recA protein with single-stranded DNA. Quantitative aspects of binding affinity modulation by nucleotide cofactors. J Mol Biol. 1985; 181(2):281-95. DOI: 10.1016/0022-2836(85)90092-0. View

Culyba M, Kubiak J, Mo C, Goulian M, Kohli R . Non-equilibrium repressor binding kinetics link DNA damage dose to transcriptional timing within the SOS gene network. PLoS Genet. 2018; 14(6):e1007405. PMC: 5999292. DOI: 10.1371/journal.pgen.1007405. View

Willetts N, Clark A, Low B . Genetic location of certain mutations conferring recombination deficiency in Escherichia coli. J Bacteriol. 1969; 97(1):244-9. PMC: 249588. DOI: 10.1128/jb.97.1.244-249.1969. View

Renzette N, Gumlaw N, Nordman J, Krieger M, Yeh S, Long E . Localization of RecA in Escherichia coli K-12 using RecA-GFP. Mol Microbiol. 2005; 57(4):1074-85. DOI: 10.1111/j.1365-2958.2005.04755.x. View

10.

Wang W, TESSMAN E . Evidence that the recA441 (tif-1) mutant of Escherichia coli K-12 contains a thermosensitive intragenic suppressor of RecA constitutive protease activity. J Bacteriol. 1985; 163(1):407-9. PMC: 219131. DOI: 10.1128/jb.163.1.407-409.1985. View

11.

Lavery P, Kowalczykowski S . Biochemical basis of the constitutive repressor cleavage activity of recA730 protein. A comparison to recA441 and recA803 proteins. J Biol Chem. 1992; 267(29):20648-58. View

12.

Little J, Mount D . The SOS regulatory system of Escherichia coli. Cell. 1982; 29(1):11-22. DOI: 10.1016/0092-8674(82)90085-x. View

13.

Nastri H, Knight K . Identification of residues in the L1 region of the RecA protein which are important to recombination or coprotease activities. J Biol Chem. 1994; 269(42):26311-22. View

14.

Madiraju M, Templin A, Clark A . Properties of a mutant recA-encoded protein reveal a possible role for Escherichia coli recF-encoded protein in genetic recombination. Proc Natl Acad Sci U S A. 1988; 85(18):6592-6. PMC: 282023. DOI: 10.1073/pnas.85.18.6592. View

15.

Knight K, Aoki K, Ujita E, McEntee K . Identification of the amino acid substitutions in two mutant forms of the recA protein from Escherichia coli: recA441 and recA629. J Biol Chem. 1984; 259(18):11279-83. View

16.

Volkert M, Margossian L, Clark A . Two-component suppression of recF143 by recA441 in Escherichia coli K-12. J Bacteriol. 1984; 160(2):702-5. PMC: 214793. DOI: 10.1128/jb.160.2.702-705.1984. View

17.

McGrew D, Knight K . Molecular design and functional organization of the RecA protein. Crit Rev Biochem Mol Biol. 2003; 38(5):385-432. DOI: 10.1080/10409230390242489. View

18.

Sandler S . Studies on the mechanism of reduction of UV-inducible sulAp expression by recF overexpression in Escherichia coli K-12. Mol Gen Genet. 1994; 245(6):741-9. DOI: 10.1007/BF00297281. View

19.

Volkert M, Hartke M . Suppression of Escherichia coli recF mutations by recA-linked srfA mutations. J Bacteriol. 1984; 157(2):498-506. PMC: 215275. DOI: 10.1128/jb.157.2.498-506.1984. View

20.

Lavery P, Kowalczykowski S . Biochemical basis of the temperature-inducible constitutive protease activity of the RecA441 protein of Escherichia coli. J Mol Biol. 1988; 203(4):861-74. DOI: 10.1016/0022-2836(88)90112-x. View