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Journal of Bacteriology, August 2003, p . 4626-4629, Vol . 185, No . 15

Competition between MutY and Mismatch Repair at A · C Mispairs In Vivo

Mandy Kim, Tiffany Huang, and Jeffrey H . Miller*

Department of Microbiology, Immunology, and Molecular Genetics and The Molecular Biology Institute, University of California, Los Angeles, California 90095

Received 3 March 2003/ Accepted 8 May 2003


   ABSTRACT

 
We show that the MutY protein competes with the MutS-dependent mismatch repair system to process at least some A · C mispairs in vivo, converting them to G · C pairs . In the presence of an increased dCTP pool resulting from the loss of nucleotide diphosphate kinase, the frequency of A · T->G · C transitions at a hot spot in the rpoB gene is 30-fold lower in a MutY-deficient derivative than in the wild type .


   TEXT

 
The MutY protein, the product of the mutY gene in Escherichia coli (18), is a glycosylase that plays an important role in the repair of oxidatively damaged DNA (11, 12) . Loss of function in both chromosomal copies of the human gene encoding MutY leads to increased susceptibility to colon cancer in humans (1, 9) . MutY removes A residues that are mispaired with 7,8-dihydro-8-oxoguanine (oxoG) (11, 13), a frequent oxidation product of DNA (4) . This removal allows repair polymerases to restore a C across from the 8-oxoG (25), allowing the MutM protein to remove the 8-oxoG (11, 13; for reviews, see references 12 and 15) . Subsequent repair synthesis restores the original G · C base pair . MutY also removes the A from A · G mispairs (2), from A · 8-oxoA mispairs (13), and, to a lesser extent, from A · C mispairs (13, 21) . Because the MutY protein does not discriminate between old and new strands, the ability to remove A from A · C mispairs may potentially immortalize mutations stemming from A · C mispairs in which A is the correct base, even though these mispairs may be substrates for correction by the MutSHL-dependent mismatch repair (MMR) system (for a review, see reference 17) . In these cases, the original A · T base pair would be converted to a G · C base pair . In this sense, the MutY protein competes with the MMR system for the processing of A · C mispairs . Figure 1 portrays the different outcomes of A · C mispairs arising from replication errors at an A · T base pair .


FIG . 1 . Competition between the MutY protein and the MutHLS MMR system . (A) Misreplication at an A · T base pair leads to an A · C mispair that is corrected by the MutHLS MMR system but converted to a G · C pair by MutY . (B) Misreplication at a G · C base pair leads to an A · C mispair that is converted back to a G · C pair by both MutY and the MMR system.

 
The E . coli rpoB/Rifr system. We decided to study the contribution of the MutY protein to A · T->G · C mutations that can arise via an A · C mispair by using the previously described E . coli rpoB/Rifr system to monitor mutations (7) . We have extended the work of others (8, 19, 22-24) to generate a system that can analyze 69 different base substitutions in the rpoB gene (7) . Recent work (20; E . Wolff, M . Kim, and J . H . Miller, unpublished data) has added several sites to this collection, so that as many as 73 different base substitutions can be monitored by analyzing E . coli Rifr mutants at 37°C (Table 1) . Of these 73 mutations, one particular A · T->G · C mutation, at bp 1547, is a hot spot in a wild-type background and a very strong hot spot in a mutS background (7, 16) . Although it is not clear what proportion of the A · T->G · C transition mutations at this site result from A · C rather than T · G mispairs, comparing the rates of mutations at this hot spot in wild-type and mutY backgrounds seems to be a straightforward way to look for effects of the presence or absence of MutY on transitions . However, G · C->T · A mutations are elevated in a mutY background, significantly increasing the Rifr mutant frequencies (17) (Table 2) . Only a small percentage of the Rifr mutants would be caused by other mutations . Thus, 13 of 15 mutations in rpoB obtained in a mutY strain result from G · C->T · A transversions (Table 1) . Therefore, we sought to increase the level of A · C mispairs by employing a strain with a defect in the ndk gene (16), which encodes the enzyme nucleotide diphosphate kinase . Nucleotide diphosphate kinase is involved in maintaining nucleotide triphosphate levels, and ndk mutant strains have 20-fold-higher levels of dCTP and 7-fold-higher levels of dGTP, as well as higher levels of spontaneous mutations (10), than do strains without a defect in the ndk gene . We have analyzed the mutator effect of ndk strains and shown that certain base substitutions and frameshifts are considerably elevated in ndk mutS double mutants, indicating that replication errors are involved (16) . Among mutations in rpoB leading to Rifr, the mutations in both ndk and ndk mutS strains predominate at the A · T->G · C hot spot at bp 1547 (16) . Because the level of mutations at this hot spot in ndk strains is high enough, we can determine whether the MutY protein is involved in generating A · T->G · C mutations at the bp 1547 hot spot by comparing the levels of mutations at this site in ndk and ndk mutY strains (for methods, see references 7 and 14) .


TABLE 1 . Distribution of mutation in rpoB

 

TABLE 2 . rpoB frequencies and notes of mutationsa

 
Distribution of mutations in rpoB. Table 1 shows the distribution of rpoB mutations in wild-type, mutS, mutY, and ndk strains and in the ndk mutY and mutS mutY double mutants . Table 2 shows some comparative mutation frequencies and mutation rates per replication . ndk strains have 43-fold-higher rates of rpoB mutations that lead to Rifr colonies than the wild type, and mutS strains have approximately a 80-fold-higher mutation rate (Table 2) than the wild type, although most of the mutations in the ndk and mutS strains are at one site (position 1547; A · T->G · C) (Table 1) . The defect in mutY has no detectable effect on the mutation rate in mutS strains (data not shown) and no effect on the mutS spectrum (Table 1; note the different sample sizes) . This lack of effect occurs because in the absence of MMR, which is the consequence of being a mutS mutant, there is no way to repair A · C mismatches, derived from A · T base pairs (Fig . 1A), regardless of the presence or absence of the MutY protein . However, in an ndk strain that is MMR proficient, the defect in the mutY gene lowers the frequency of mutations in rpoB 2.5- to 3-fold (Table 2) and, most importantly, virtually eliminates the position 1547 A · T -> G · C hot spot from the mutational spectrum (Table 1) . In the absence of this hot spot, the next most frequent mutation, A · T->T · A at position 443, now becomes the most frequent mutation and appears as a new hot spot . Figure 2 incorporates the mutation frequencies into the comparison of the ndk and ndk mutY spectra and shows that MutY-deficient derivatives of ndk strains have 30-fold-lower levels of A · T->G · C mutations at position 1547 in rpoB . (The apparent severalfold increase of A · T->T · A mutations at position 443 may not be significant, because of the small sample size of these mutations at position 443 in the distribution for the ndk mutants.) This finding demonstrates an in vivo effect of MutY on increasing transition mutations at certain A · T base pairs due to its ability to remove A from A · C mispairs and represents an example of a repair enzyme actually being involved in creating mutations under certain conditions . The effect of MutY on lowering A · T->G · C transversions in a mutT background has been described previously (6, 26), as has a 4.6-fold decrease in A · T->G · C mutations at one site in trpA (6) .


FIG . 2 . Comparative mutation frequencies in ndk and ndk mutY strains . The frequencies of mutations in the rpoB gene at five different sites are shown for both ndk (white bars) and ndk mutY (black bars) backgrounds (see also Tables 1 and 2) . The five sites (left to right) are as follows: 1, A · T->T · A at bp 443; 2, A · T->T · A at bp 1532; 3, A · ->G · C at bp 1532; 4, A · T->G · C at bp 1534; 5, A · T->G · C at bp 1547.

 


   ACKNOWLEDGMENTS

 
This work was supported by a grant from the National Institutes of Health (grant ES0110875) .


   FOOTNOTES

 
* Corresponding author . Mailing address: Department of Microbiology, Immunology, and Molecular Genetics, UCLA, 405 Hilgard Ave., Los Angeles, CA 90095 . Phone: (310) 825-8460 . Fax: (310) 206-3088 . E-mail: jhmiller{at}mbi.ucla.edu .


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