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Mismatch Repair Protein Mutl Becomes Limiting During Stationary-Phase Mutation

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Mismatch Repair Protein Mutl Becomes Limiting During Stationary-Phase Mutation Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Mismatch repair protein MutL becomes limiting during stationary-phase mutation Reuben S. Harris,1 Gang Feng,2 Kimberly J. Ross,1 Roger Sidhu, Carl Thulin, Simonne Longerich, Susan K. Szigety, Malcolm E. Winkler,2 and Susan M. Rosenberg1,3 Department of Biochemistry, University of Alberta Faculty of Medicine, Edmonton, Alberta T6G 2H7 Canada; 2Department of Microbiology and Molecular Genetics, University of Texas Houston Medical School, Houston, Texas 77030-1501 USA Postsynthesis mismatch repair is an important contributor to mutation avoidance and genomic stability in bacteria, yeast, and humans. Regulation of its activity would allow organisms to regulate their ability to evolve. That mismatch repair might be down-regulated in stationary-phase Escherichia coli was suggested by the sequence spectrum of some stationary-phase (‘‘adaptive’’) mutations and by the observations that MutS and MutH levels decline during stationary phase. We report that overproduction of MutL inhibits mutation in stationary phase but not during growth. MutS overproduction has no such effect, and MutL overproduction does not prevent stationary-phase decline of either MutS or MutH. These results imply that MutS and MutH decline to levels appropriate for the decreased DNA synthesis in stationary phase, whereas functional MutL is limiting for mismatch repair specifically during stationary phase. Modulation of mutation rate and genetic stability in response to environmental or developmental cues, such as stationary phase and stress, could be important in evolution, development, microbial pathogenicity, and the origins of cancer. [Key Words: Mismatch repair; MutL; mutation; adaptive mutation; genetic instability; evolution; stationary phase] Received April 4, 1997; revised version accepted July 18, 1997. In Escherichia coli mismatch repair is the single largest Four proteins play critical roles in mismatch repair in contributor to avoidance of mutations due to DNA poly- E. coli (for review, see Modrich 1991). MutS binds to merase errors in replication (Radman 1988; Modrich DNA base mismatches, to insertion/deletion single- 1991). Mismatch repair also promotes genetic stability strand loops of four or fewer nucleotides, which are in- by editing the fidelity of genetic recombination and termediates in frameshift mutation, and probably also to transposon excision, and by the involvement of its com- sites of DNA damage (Mello et al. 1996). MutL interacts ponent proteins in transcription-coupled DNA repair with MutS after mismatch binding and is thought to and very-short-patch repair (Radman 1988; Modrich coordinate MutS with MutH. MutH endonuclease then 1991; Lieb and Shehnaz 1995; Mellon and Champe 1996). nicks the unmethylated (new) DNA strand of a nearby The mismatch repair proteins are highly conserved hemimethylated GATC sequence. GATCs are hemi- throughout evolution and appear to play roles in simple methylated during DNA replication because the new and complex eukaryotes similar to those that they play strand is transiently unmethylated. MutU helicase en- in bacteria (Reenan and Kolodner 1992; Modrich 1994; ters DNA at the single-strand nick and displaces the Baker et al. 1995, 1996; de Wind et al. 1995; Datta et al. nicked strand, which may or may not be degraded during 1996; Hunter et al. 1996; Kolodner 1996). These proteins displacement (see R.S. Harris, K.J. Ross, M.-J. Lombardo, act on incorrectly paired and unpaired bases in DNA that and S.M. Rosenberg, unpubl.). DNA polymerase III then arise via DNA synthesis errors, recombination of di- resynthesizes the DNA, thus correcting the sequence. In verged sequences, and DNA damage. In all of these cir- recombination, the mismatches or small loops are cumstances, mismatch repair enforces genetic stability. caused by formation of heteroduplex DNA between non- The consequences of failing to maintain this enforce- identical sequences rather than by replication errors as ment are profound for speciation (Rayssiguier et al. 1989; just described. Radman and Wagner 1993; Matic et al. 1995; Hunter et Although central to maintenance of genetic stability, al. 1996; Zahrt and Maloy 1997) and for formation of little is known about whether the mismatch repair sys- cancers (Modrich 1994, 1995; Kolodner 1996). tem might be regulated (but see Stahl 1988; Rayssiguier et al. 1989; Hastings and Rosenberg 1992; Rosenberg 1994, 1997; Longerich et al. 1995; Rosenberg et al. 1995, 1Present address: Department of Molecular and Human Genetics, Baylor 1996 for hypotheses). If mismatch repair were regulated, College of Medicine, Houston, Texas 77030 USA 3Corresponding author. then cells would regulate their potential to evolve. Two E-MAIL [email protected]; FAX: (713) 798-5386. lines of work from E. coli have converged on the first 2426 GENES & DEVELOPMENT 11:2426–2437 © 1997 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/97 $5.00 Downloaded from genesdev.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press MutL becomes limiting during stationary phase evidence in any organism suggesting that mismatch re- protein, specifically during the differentiated state of sta- pair proteins might be regulated and, more specifically, tionary phase. This provides the first evidence in any have suggested the down-regulation of mismatch repair organism that mismatch repair function is not constitu- during the differentiated states of stationary phase and tive but, rather, can be modulated by cell physiology and nutritional stress. differentiation. Environmental change could promote ge- First, stationary-phase reversions of a lac +1 frameshift netic change by this route. mutation in E. coli (Cairns and Foster 1991) appear to be DNA polymerase errors that escape mismatch repair. Results The stationary-phase reversion mechanism in the lac frameshift assay system is distinct from growth-depen- The term stationary-phase mutation is used here to refer dent Lac reversion (Rosenberg 1994, 1997; Rosenberg et to what has also been called ‘‘adaptive mutation’’ (for al. 1995, 1996) in that the former includes homologous review, see Foster 1993). This process occurs in stressed, recombination (Harris et al. 1994, 1996; Foster et al. starving cells and so may reflect stress responses in gen- 1996) and produces mutations with a highly distinctive eral. In the assay system used here—reversion of a lac DNA sequence spectrum, mostly single-base deletions frameshift mutation in E. coli—the stationary-phase mu- in small mononucleotide repeats (Foster and Trimarchi tations are also mechanistically distinct from growth- 1994; Rosenberg et al. 1994). This mutation spectrum is dependent reversions of the same allele. Unlike growth- different from growth-dependent reversions of the same dependent Lac reversion, the stationary-phase mutation allele (Foster and Trimarchi 1994; Rosenberg et al. 1994) mechanism (1) requires homologous recombination but is identical to growth-dependent reversions in mis- (Harris et al. 1994, 1996; Foster et al. 1996); (2) includes match repair–null mutant strains (Longerich et al. 1995). DNA double-stand breaks that are implicated as a mo- Thus, depressed mismatch repair could be responsible lecular intermediate in the mutagenesis (Harris et al. for the unique stationary-phase mutation spectrum. 1994); and (3) produces mutations that are nearly all Most stationary-phase mutants are not heritably mis- single-base deletions in small mononucleotide repeats, match repair-defective (Longerich et al. 1995) [although whereas growth-dependent Lac reversions are heterog- some are (Torkelson et al. 1997)], indicating that any loss eneous (Foster and Trimarchi 1994; Rosenberg et al. of mismatch repair during stationary-phase mutation 1994). Furthermore, (4) this unique sequence spectrum is must be transient. Such transient loss could occur either identical to that observed in mismatch repair-defective by down-regulation of the mismatch repair system or by cells, implying failure of mismatch repair in stationary- a block at the DNA level, for example, either by under- phase mutation (Longerich et al. 1995); and (5) although or overmethylation of DNA sites required for operation this question has not been addressed for the growth-de- of this methyl-directed repair system (Longerich et al. pendent mutations, the stationary-phase mutations have 1995). been shown to occur genome-wide, in a hypermutable Second, the recent discovery that MutS and MutH subpopulation of the stressed cells (Torkelson et al. mismatch repair protein levels decrease in stationary 1997). This specific mutation mechanism is called re- phase and starving bacterial cells appears to support the combination-dependent stationary-phase mutation. It is hypothesis of down-regulation of mismatch repair at the proposed to occur by DNA polymerase errors occurring protein level (Feng et al. 1996). However, although MutS during DNA synthesis primed by recombinational and MutH protein levels decrease during stationary strand-exchange intermediates, which form at DNA phase and starvation, so does DNA replication. There- double-strand breaks, in cells with attenuated mismatch fore, it is possible that the proper ratio of these proteins repair (for review, see Rosenberg 1994, 1997; Rosenberg to replication errors is preserved, leaving mismatch re- et al. 1995, 1996) or in DNA substrates that are refrac- pair functional in stationary-phase, starving cells.
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