Genetics, Vol. 150, 533-542, October 1998, Copyright © 1998

Mechanism and Control of Interspecies Recombination in Escherichia coli. I. Mismatch Repair, Methylation, Recombination and Replication Functions

Snjezana Stambuka and Miroslav Radmana
a Laboratoire de Mutagénèse, Institut Jacques Monod, 75251-Paris Cedex 05, France

Corresponding author: Snjezana Stambuk, Laboratoire de Mutagénèse, Institut J. Monod, Université Paris 7, 2, place Jussieu, 75251, Paris, France., This email address is being protected from spambots. You need JavaScript enabled to view it. (E-mail).

Communicating editor: P. J. PUKKIL



A genetic analysis of interspecies recombination in Escherichia coli between the linear Hfr DNA from Salmonella typhimurium and the circular recipient chromosome reveals some fundamental aspects of recombination between related DNA sequences. The MutS and MutL mismatch binding proteins edit (prevent) homeologous recombination between these 16% diverged genomes by at least two distinct mechanisms. One is MutH independent and presumably acts by aborting the initiated recombination through the UvrD helicase activity. The RecBCD nuclease might contribute to this editing step, presumably by preventing reiterated initiations of recombination at a given locus. The other editing mechanism is MutH dependent, requires unmethylated GATC sequences, and probably corresponds to an incomplete long-patch mismatch repair process that does not depend on UvrD helicase activity. Insignificant effects of the Dam methylation of parental DNAs suggest that unmethylated GATC sequences involved in the MutH-dependent editing are newly synthesized in the course of recombination. This hypothetical, recombination-associated DNA synthesis involves PriA and RecF functions, which, therefore, determine the extent of MutH effect on interspecies recombination. Sequence divergence of recombining DNAs appears to limit the frequency, length, and stability of early heteroduplex intermediates, which can be stabilized, and the recombinants mature via the initiation of DNA replication.

HOMOLOGOUS genetic recombination is required for DNA repair and for meiotic crossovers involved in chromosome disjunction (KUCHERLAPATI and SMITH 1988 Down). However, crossovers between interspersed repeated sequences cause deleterious chromosomal rearrangements (for review see RADMAN 1991 Down). A balance between the positive and negative effects of homologous recombination is kept by cellular mechanisms that control its frequency and fidelity. The key role of homologous recombination in the repair of DNA probably sets the limits to the extent of negative control of recombination. For example, a cellular Rec- phenotype could be favored to avoid chromosomal rearrangements, but it would lead to a great disadvantage because of the deficiency in DNA repair. The solution to this particular problem appears to be provided by the high fidelity of homologous recombination (i.e., its strict requirement for sequence identity): DNA repair can proceed by unrestricted precise recombination between the identical sister chromatids, whereas the nonidentity of repeated sequences prevents their recombination (RAYSSIGUIERet al. 1989 Down; PETIT et al. 1991 Down; RADMAN 1991 Down; ABDULKARIM and HUGHES 1996 Down).

Thus, homologous genetic recombination is largely controlled at the DNA substrate level by the degree and length of sequence identity shared by the two recombining DNAs (for review see RADMAN 1991 Down). The decrease in DNA homology through sequence divergence is much more efficient in preventing recombination than is the decrease in the length of homology (SHEN and HUANG 1986 Down). Even a low divergence, e.g., 1% or less, can severely inhibit homologous recombination in bacteria, yeast, and mammalian cells (DE WINDet al. 1995 Down; DATTAet al. 1996 Down, DATTAet al. 1997 Down; VULICet al. 1997 Down; ZAHRT and MALOY 1997 Down). This high fidelity of genetic recombination is caused not only by the intrinsic properties of recombination enzymes, but also by the editing of recombination by the mismatch repair system, the same system that controls also the fidelity of DNA replication through the correction of base pair mismatches caused by replication errors (for review see RADMAN and WAGNER 1993ADown; MODRICH and LAHUE 1996 Down).

Deficiency in any of the four mismatch repair proteins, MutS, MutL, MutH, and UvrD (MutU), has equal effect on the correction of DNA replication errors, reflecting the requirement of all four proteins in the successful error correction process (for review see RADMAN and WAGNER 1986 Down). This is generally not the case for the editing of homologous DNA recombination, where the effect of specific mut gene mutations depends on the nature of recombination substrates and events (PETITet al. 1991 Down; ABDULKARIM and HUGHES 1996 Down). Interspecies recombination between Salmonella and Escherichia in conjugational and transductional crosses is increased ~1000-fold by mutS and/or mutL mutations, ~20-fold by mutH, and only about fivefold by a mutU mutation (RAYSSIGUIERet al. 1989 Down; see Table 2).


We have been studying gene exchange between two related species, Escherichia coli and Salmonella typhimurium, mediated by homologous recombination enzymes as a model system for the definition of the genetic barriers at the molecular level (RAYSSIGUIERet al. 1989 Down; MATICet al. 1995 Down; VULICet al. 1997 Down). The principal genetic barrier among enterobacteria is the recombinational barrier, whose structural element is the genomic sequence divergence and whose enzymatic element is the mismatch repair system. So far, the basic molecular rules for this interspecies recombination appear to apply also to other homologous recombination systems involving similar sequences (homeologous recombination) in bacteria, yeast, and mammals (RADMAN and WAGNER 1993ADown; DE WINDet al. 1995 Down; DATTAet al. 1996 Down, DATTAet al. 1997 Down; VULICet al. 1997 Down). Although the editing of homeologous strand exchange by the MutS and MutL proteins has been reproduced in vitro (WORTH et al. 1994 Down), the detailed mechanism of the editing of recombination by the mismatch repair system remains obscure.

This is a study of the roles of DNA methylation and MutS/L vs. MutH functions in recombinational editing. By studying the fidelity of interspecies recombination, we found that (i) this recombination often involves DNA synthesis initiated by a pairing between 16% (on average) diverged parental sequences and that requires the PriA primosome function and some RecF functions and (ii) editing of recombination by mismatch repair proteins occurs by two mechanisms: one is MutH independent and the other is MutH dependent.




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