(NAS Colloquium) Links Between Recombination and Replication: Vital Roles of Recombination

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Colloquium on Links Between Recombination and Replication: Vital Roles of Recombination

Colloquium

Stationary-phase mutation in the bacterial chromosome: Recombination protein and DNA polymerase IV dependence

Harold J.Bull*^†^‡, Mary-Jane Lombardo*^†, and Susan M.Rosenberg*^§¶^||

Departments of *Molecular and Human Genetics, ^¶Biochemistry, and ^§Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030–3411

Several microbial systems have been shown to yield advantageous mutations in slowly growing or nongrowing cultures. In one assay system, the stationary-phase mutation mechanism differs from growth-dependent mutation, demonstrating that the two are different processes. This system assays reversion of a lac frameshift allele on an F′ plasmid in Escherichia coli. The stationary-phase mutation mechanism at lac requires recombination proteins of the RecBCD double-strand-break repair system and the inducible errorprone DNA polymerase IV, and the mutations are mostly −1 deletions in small mononucleotide repeats. This mutation mechanism is proposed to occur by DNA polymerase errors made during replication primed by recombinational double-strand-break repair. It has been suggested that this mechanism is confined to the F plasmid. However, the cells that acquire the adaptive mutations show hypermutation of unrelated chromosomal genes, suggesting that chromosomal sites also might experience recombination protein-dependent stationary-phase mutation. Here we test directly whether the stationary-phase mutations in the bacterial chromosome also occur via a recombination protein- and pol IV-dependent mechanism. We describe an assay for chromosomal mutation in cells carrying the F′ lac. We show that the chromosomal mutation is recombination protein- and pol IV-dependent and also is associated with general hypermutation. The data indicate that, at least in these male cells, recombination protein-dependent stationary-phase mutation is a mechanism of general inducible genetic change capable of affecting genes in the bacterial chromosome.

Escherichia coli | adaptive mutation | SOS response | DNA repair

Adaptive (or stationary-phase) mutation is a collection of phenomena in which mutations occur in populations of stressed, nongrowing, or slowly growing cells, and at least some of these mutations allow growth (reviewed by refs. 1–4). Stationary-phase mutation mechanisms may be important in development of antibiotic resistance mutations (5), phase variation in bacterial pathogens (3), and colonization of new bacterial hosts (6). Stationary-phase mutation mechanisms also may provide models for mutational escape of growth control, such as in oncogenesis, tumor progression, and resistance to chemotherapeutic drugs (7), and imply that genetic changes that fuel evolution may be accelerated during stress. Adaptive mutational processes contrast with the spontaneous mutation paradigm of Luria and Delbrück (8) in which mutations arise in growing cells, before cells are exposed to a selective environment, and more or less randomly in genomes. [These are the only mutations observed when the selection for mutants is lethal (reviewed in refs. 1 and 3).] It has been important to understand whether adaptive mutational processes represent departures in mechanism from spontaneous growth-dependent mutational processes, or whether they are merely growth-dependent mutations occurring in cell populations in which growth is difficult to measure. Stationary-phase mutations have been demonstrated to form via mechanisms unlike mutation in growing cells (and so, demonstrably, to be different processes) in only three experimental systems: (i) an assay that measures transposon-mediated deletions in Escherichia coli (9–11), (ii) an assay for substitution mutations in old E. coli colonies (12, 13), and (iii) the lac frameshift reversion assay in E. coli (14). The lac system measures reversion of a lac+1 frameshift mutation carried on an F′ conjugative plasmid in E. coli cells starved on lactose medium (14). The stationary-phase mutation mechanism at work in the lac system is the best characterized of any stationary-phase mutation mechanism and is the focus of this report.

The stationary-phase mutations at lac can be distinguished from growth-dependent Lac⁺ reversions as follows. The stationary-phase mutations occur in Lac− starving cells after exposure to lactose medium (15) at high frequency, accumulating over time (14). Unlike growth-dependent mutations, these require homologous recombination and double-strand break repair (DSBR) proteins RecA, RecBC, and RuvA, RuvB, and RuvC (16–18), implicating double-strand DNA breaks or double-strand ends as intermediates in mutation. F′ transfer functions, but not actual transfer, are required (19–21), suggesting that some aspect of the transfer process promotes mutation. An intact SOS response to DNA damage also is required for efficient stationary-phase mutation (14, 22), most of which requires the SOS error-prone DNA polymerase IV, encoded by dinB (23). The major replicative polymerase, DNA pol III, also has been implicated (23–25). The adaptive mutations are proposed to result from DNA polymerase errors accrued during replication primed by DSBR recombination (16) (although other models are possible; ref. 3 and discussed below). Finally, the cells that become Lac⁺, but not their similarly starved Lac⁻ neighbors, carry high frequencies of additional, unselected mutations (26–29), but are not heritably mutator (26, 27, 30, 31). This finding suggests that some or all of the adaptive mutants arise in a transiently hypermutable subpopulation of cells (as proposed originally for recombination-independent stationary-phase mutations; refs. 32 and 33, and see refs. 29, 34, and 35, and below for further discussion regarding the Lac system). The additional

This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA.

Abbreviations: DSB, double-strand break; DSBR, DSB repair; LBH, Luria-Bertani-Herskowitz medium.

^†	H.J.B. and M.-J.L. contributed equally to this work.
^‡	Present address: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E5.
^\|\|	To whom reprint requests should be addressed at: Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Room S809A, Mail Stop BCM225, Houston, TX 77030–3411. E-mail: smr@bcm.tmc.edu.

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