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

Copyright1998 by the Genetics Society of America

Mechanism and Control of Interspecies Recombination in Escherichia coli.

I. Mismatch Repair, Methylation, Recombination and Replication Functions

Snjezˇana Sˇtambuk and Miroslav Radman

Laboratoire de Mutage´ne`se, Institut Jacques Monod, 75251-Paris Cedex 05, France

Manuscript received March 16, 1998 Accepted for publication July 6, 1998

ABSTRACT

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

recom-bination 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.

H

OMOLOGOUS genetic recombination is re- Thus, homologous genetic recombination is largely controlled at the DNA substrate level by the degree and quired for DNA repair and for meiotic crossovers

involved in chromosome disjunction (Kucherlapati length of sequence identity shared by the two recombin-ing DNAs (for review seeRadman1991). The decrease and Smith1988). However, crossovers between

inter-in DNA homology through sequence divergence is spersed repeated sequences cause deleterious

chromo-much more efficient in preventing recombination than somal rearrangements (for review seeRadman 1991).

is the decrease in the length of homology (Shen and A balance between the positive and negative effects of

Huang1986). Even a low divergence, e.g., 1% or less, homologous recombination is kept by cellular

mecha-can severely inhibit homologous recombination in bac-nisms that control its frequency and fidelity. The key

teria, yeast, and mammalian cells (de Windet al. 1995; role of homologous recombination in the repair of DNA

Dattaet al. 1996, 1997;Vulicet al. 1997; Zahrtand probably sets the limits to the extent of negative control

Maloy1997). This high fidelity of genetic recombina-of recombination. For example, a cellular Rec2

pheno-tion is caused not only by the intrinsic properties of type could be favored to avoid chromosomal

rearrange-recombination enzymes, but also by the editing of re-ments, but it would lead to a great disadvantage because

combination by the mismatch repair system, the same of the deficiency in DNA repair. The solution to this

system that controls also the fidelity of DNA replication particular problem appears to be provided by the high

through the correction of base pair mismatches caused fidelity of homologous recombination (i.e., its strict

re-by replication errors (for review see Radman and quirement for sequence identity): DNA repair can

pro-Wagner1993a;ModrichandLahue1996). ceed by unrestricted precise recombination between

Deficiency in any of the four mismatch repair proteins, the identical sister chromatids, whereas the nonidentity

MutS, MutL, MutH, and UvrD (MutU), has equal effect of repeated sequences prevents their recombination

on the correction of DNA replication errors, reflecting (Rayssiguier et al. 1989; Petit et al. 1991; Radman

the requirement of all four proteins in the successful 1991; AbdulkarimandHughes1996).

error correction process (for review seeRadman and Wagner1986). This is generally not the case for the editing of homologous DNA recombination, where the M. Radman dedicates this paper to Jan Drake, whose editorial policy _{effect of specific mut gene mutations depends on the} and sharp editorial pencil rejuvenated Genetics.

nature of recombination substrates and events (Petitet

Corresponding author: Snjezˇana Sˇtambuk, Laboratoire de

Mutage´-al. 1991;AbdulkarimandHughes1996). Interspecies

ne`se, Institut J. Monod, Universite´ Paris 7, 2, place Jussieu, 75251,

Paris, France. E-mail: [email protected] recombination between Salmonella and Escherichia in

gants in crosses involving SA965 and SA977 donors. Resuspen-conjugational and transductional crosses is increased

sion of filters on which crosses with SU573 and SA534 donors

z1000-fold by mutS and/or mutL mutations, z20-fold

were plated on similar plates, but to select for leucine prototro-by mutH, and only about fivefold prototro-by a mutU mutation _{phy. In interspecies crosses where the recipients used were S.} (Rayssiguieret al. 1989; see Table 2). _{typhimurium and donor E. coli PK3, selection was on M63}

mini-mal medium containing glucose (0.4%), thiamine (30 mg/ We have been studying gene exchange between two

ml), isoleucine, and valine (100mg/ml), and Streptomycin related species, Escherichia coli and Salmonella

typhimu-(100 mg/ml) to counterselect donor cells. Colonies were rium, mediated by homologous recombination enzymes

counted after 4 days or more if scoring for the frequency of as a model system for the definition of the genetic barri- _{recombinants of priA cells was performed. In an intraspecies} ers at the molecular level (Rayssiguier et al. 1989;

cross where the donor used was PK3, selection was performed

Matic et al. 1995; Vulic et al. 1997). The principal _{for arginine marker.}

To minimize the appearance of suppressor mutations be-genetic barrier among enterobacteria is the

recombina-cause of the mutator effect of mutS and mutH recipients used, tional barrier, whose structural element is the genomic

the priA and recF strains were grown in M63 minimal medium sequence divergence and whose enzymatic element is _{containing glucose (0.4%,McPhee1993), histidine, leucine,} the mismatch repair system. So far, the basic molecular _{proline, arginine, threonine (100mg/ml), and thiamine (30} rules for this interspecies recombination appear to apply mg/ml) until their midexponential phase of regrowth, and were then agitated for 3 hr in rich medium before conjugation. also to other homologous recombination systems

involv-Recombination frequency is expressed as the number of re-ing similar sequences (homeologous recombination) in

combinants per Hfr donor. Values represent the means and bacteria, yeast, and mammals (Radman and Wagner

standard deviations of the recombination frequencies ob-1993a; de Wind et al. 1995; Datta et al. 1996, 1997;

tained in independent crosses.

Vulicet al. 1997). Although the editing of homeologous strand exchange by the MutS and MutL proteins has been reproduced in vitro (Worthet al. 1994), the

de-RESULTS AND DISCUSSION tailed mechanism of the editing of recombination by

MutS vs. MutH effects on interspecies recombination:

the mismatch repair system remains obscure.

Presumably because of their mismatch recognition and This is a study of the roles of DNA methylation and

binding activities (for review seeModrichandLahue MutS/L vs. MutH functions in recombinational editing.

1996), the MutS and MutL functions appear indispens-By studying the fidelity of interspecies recombination,

able for all modes of the editing of DNA recombination we found that (i) this recombination often involves DNA

and replication (this paper and unpublished results synthesis initiated by a pairing between 16% (on

aver-from our laboratory). The roles of MutH and UvrD in age) diverged parental sequences and that requires the

the editing of recombination are poorly understood. PriA primosome function and some RecF functions and

The key role of MutH in the mismatch repair process (ii) editing of recombination by mismatch repair

pro-is to produce a single-strand scpro-ission (nick) 59 to an teins occurs by two mechanisms: one is MutH

indepen-unmethylated GATC sequence. This MutH activity is dent and the other is MutH dependent.

somehow stimulated by the binding of MutS protein to a nearby mismatch (mispaired or unpaired bases in duplex DNA) followed by binding of MutL. The uvrD MATERIALS AND METHODS

gene encodes DNA helicase II, whose role in mismatch Bacterial strains and plasmids: The bacterial strains used _{repair is supposedly to peel off the nicked strand in the} are listed in Table 1. Figure 1 shows the position and direction

direction of the mismatch and expose the free single of the transfer of the Hfr strains used in this study. SA965,

strand for destruction by single-strand-specific nucleases SA977, SA534, and SU573 are S. typhimurium Hfr, while PK3

is E. coli Hfr. Plasmid pDDM6, which carries the gene coding (for review seeModrichandLahue 1996).

for the Dam methylase enzyme (Guhaand Guschlbauer

To determine the epistatic relationships of such muta-1992) under the control of the tac promoter, was propagated _{tions, we have studied the effect of a series of single} in mut1and mutH (AB1157) strains in the presence of 3 mm

and double mutations that affect mismatch repair and/ IPTG to induce Dam methylase.

or recombination machinery. Such analysis is useful for Conjugational crosses: Donor and recipient strains were

grown in rich medium or minimal medium where indicated. pathway assignments of genes that control the fidelity M63 minimal medium contained glucose (0.4%), histidine, _{of genetic recombination. A large number of mutations} leucine, proline, arginine, threonine (100mg/ml), and thia- _{studied makes the epistasis analysis notoriously difficult.} mine (30 mg/ml). Log-phase bacteria were grown to 2–43

Therefore, we chose to express the effects of mutS and 108_{cells/ml, mixed in a 1:1 Hfr:F}2_{ratio, and filtered through}

mutH mutations in different mutational backgrounds a 0.45-mm pore size filter (Schleicher & Schuell, Keene, NH)

that was incubated on prewarmed, rich medium agar for 40 (Table 2). Because the MutS function is involved in all or 60 min. Filters were resuspended in 1022_mMgSO

4, and whereas MutH is involved in only some aspects of editing

bacteria were separated by vortexing. Bacteria were plated on _{of recombination by mismatch repair, the ratio of MutS} M63 minimal medium supplemented with histidine, leucine,

to MutH effects shows the extent of MutH-independent proline, and threonine (100mg/ml), thiamine (30mg/ml),

editing (Table 2). glucose (0.4%), and nalidixic acid (40mg/ml) to

TABLE 1

E. coli and S. typhimurium strains and plasmids used in this study

Strain or plasmid Relevant genotype Source of strain or allele

FR186 mutH471::Tn5 Panget al. (1985)

FR188 mutS215::Tn10 G. Walker’s collection

FR189 uvrD260::Tn5 Panget al. (1985)

MG1655 recD::Tn10 D. Touati’s collection

AQ8845 priA2::kan Masaiet al. (1994)

JC10990 recF::Tn3 A. J. Clark’s collection

ES548 mutH thy1 VaccaroandSiegel(1977)

GC2277 sfiAII pyrD:: Tn5 R. D’ari’s collection

GY4375 dam13::Tn9 Marinuset al. (1983)

TT11691a _{dam-101::Tn10 K. E. Sanderson’s collection}

FR146a, c _mut1_dam1 _{K. E. Sanderson’s collection}

FR147a, c _{mutH 101::Tn5} Rayssiguieret al. (1989)

FR826a, c _{dam-101::Tn10 This study}

FR827a, c _{mutH101::Tn5 dam-101::Tn10 This study}

FR266b _recA1_mut1 Maticet al. (1995)

FR828b _{mutH471::Tn5 This study}

FR559b _mutS201::Tn5 Matic_{et al. (1995)}

FR829b _{uvrD260::Tn5 This study}

FR830b _{uvrD260::Tn5 mutH thy}1 _{This study}

FR831b _{uvrD260::Tn5 mutS215::Tn10 This study}

FR483b _recD1009 Matic_{et al. (1995)}

FR832b _{recD::Tn10 mutH thy}1 _{This study}

FR470b _{recD1009 mutS215::Tn10 Maticet al. (1995)}

FR833b DrecG263 KmR Maticet al. (1995)

FR834b DrecG263 KmR_{mutH thy}1 _{This study}

FR835b DrecG263 KmR_{mutS215::Tn10 This study}

FR836b _ruvA60::Tn10 Maticet al. (1995)

FR837b _{ruvA60::Tn10 mutH471::Tn5 This study}

FR838b _{ruvA60::Tn10 mutS201::Tn5} Maticet al. (1995)

FR358b _{lexA1 malB::Tn9} Matic_{et al. (1995)}

FR839b _{lexA1 malB::Tn9 mutH471::Tn5 This study}

FR840b _{lexA1 malB::Tn9 mutS201::Tn5} Maticet al. (1995)

FR841b _{recF::Tn3 This study}

FR842b _{recF::Tn3 mutH471::Tn5 This study}

FR843b _{recF::Tn3 mutS215::Tn10 This study}

FR844b _{priA2::kan sf iAII This study}

FR845b _{priA2::kan mutH thy}1_{sf iAII This study}

SA977a _{leuBCD39 ara7 P22}R_RifR _{K. E. Sanderson’s collection}

SA965a _{leuBCD39 ara7 Rif}R _{K. E. Sanderson’s collection}

SU573a _{purC7 K. E. Sanderson’s collection}

SA534a _{serA13 rfa-3058 K. E. Sanderson’s collection}

PK3 lacY thi- leuB6 azi 15 tonA21 supE44 RifR Kahn (1968)

PK3 dam lacY thi- leuB6 azi15 tonA21 supE44 dam13:: Tn9 This study

pDDM6 Dam methylase GuhaandGuschlbauer(1992)

All constructions were done by P1 transduction. a_{Salmonella typhimurium LT2 strains.}

b_{AB1157 Nal}R_{(thr-1 leuB6 proA2 his4 thi-1 argE3 lacY1 galK2 ara14 xy15 mtl-1 tsx-33 str31 supE44).}

c_{SL4213 [galE496 metA22 metE551 rpsl120 xyl-404 (Fels2) H1b n}ml H2-enx ilv-452 hsdSA29 hsdL6].

decreased in uvrD, recD, recG, and recF mutants. LexA1 recombination frequencies are similar in the lexA1, recF, and priA mutants, it may be that the effects of RecF and (a dominant mutation preventing the induction of SOS

response), uvrD, recD, recG, and recF show a pronounced PriA functions and/or of some components active in the same pathway are inducible because of the SOS decrease in the MutS effect, whereas priA, recF, and lexA1

also show a decreased effect of MutH. The mutH effect response that is activated in E. coli during the course of conjugation with Salmonella (Maticet al. 1995). The on interspecies recombination is increased from z

20-fold in the wild type toz120-fold in recD, 50-fold in uvrD, significance of these findings will be discussed below.

MutH-dependent editing of interspecies

in the article byRayssiguieret al. (1989), which were performed too long ago. The results in Table 4 show that the effect of MutH on interspecies recombination is not affected by the methylation status of parental DNAs in a way that could be expected from the known roles of the MutH protein and unmethylated GATC sequences. For example, in the interspecies cross be-tween S. typhimurium F2(Dam2) and E. coli, Hfr (Dam1 or Dam2), one would expect the highest effect of MutH-dependent rejection of the Hfr DNA, i.e., the highest ratio of recombination in mutH compared with mutH1 crosses. In the total absence of Dam methylation, even the inactivation of mismatched intermediates by the MutS, L, and H activities can be expected (Doutriaux et al. 1986). However, this was not found: when both parents are Dam1, the MutH effect on recombination is 10-fold (line 1 vs. line 3), as compared to 13-fold when only the Hfr DNA is unmethylated (line 5 vs. line 7); when both DNAs are unmethylated, the MutH effect

Figure 1.—The schematic representation of the position

is also 10-fold (line 6 vs. line 8). Note that independently and direction of transfer of Hfr donors used in this study (see

materials and methods). E. coli Hfr strain is PK3, while _{of the donor’s DNA methylation, recombination}

fre-S. typhimurium Hfr strains are SA977, SA965, SA534, and _{quency is about five times higher in dam recipients,}

SU573.

presumably because of their constitutive expression of SOS response (PetersonandMount1993).

PriA, recF, and lexA1 mutations affect

MutH-depen-tion requires unmethylated GATC sequences:Methyla- _{dent editing of interspecies recombination:Because the} tion of the adenine moiety in the GATC sequence to _{MutH-dependent editing of recombination} intermedi-6-methyl-adenine by the Dam methylase prevents the _{ates requires unmethylated GATC sequences (Table 3),} MutH activity and, thus, prevents mismatch repair (for _{which are probably not carried by the parental DNAs} review seeModrichandLahue1996). However,

inter-(Table 4), it may be that a DNA synthesis mechanistically nal free ends, such as nicks, can substitute for both the _{associated with genetic recombination produces such} GATC sequence and MutH function (Lahueet al. 1987;

sequences. The functions of two proteins are supposed La¨ ngle-Roualtet al. 1987).

to be involved in both DNA recombination and replica-Table 3 shows that the overexpression of Dam methyl- _{tion: the primosome assembly protein PriA, which is} ase (in the F2cells) during the course of S. typhimurium _{apparently required for replication dependent} recombi-Hfr3E. coli F2conjugational crosses increases interspe- _{nation (}Kogomaet al. 1996;Sandleret al. 1996), and cies recombination to the same extent as the mutH muta- _{the RecF recombination protein, which was claimed to} tion. Furthermore, Dam overexpression has no effect _{be required for resumption of replication forks} dis-on recombinatidis-on in a mutH mutant (Table 3). This _{rupted by radiation damage to DNA (}Courcelleet al. result suggests that the MutH-dependent editing of re- _{1997). PriA protein is hypothesized to recognize sites} combination requires unmethylated GATC sequences _{for loading of DnaB helicase onto the single-stranded} in one or both strands of the DNA heteroduplex, just _{DNA within a D loop structure produced by the invading} as in the case of standard mismatch repair. _{DNA strand and, in that way, permit the primosome} What is the origin of unmethylated GATC sequences _{assembly system to initiate replication from the invading} in recombination: parental Hfr DNA, parental newly _{DNA strand (}Kogoma 1996;McGlynnet al. 1997). synthesized F2DNA, or some particular DNA synthesis _{If the MutH editing step of recombination requires} associated with the recombination process? _{de novo DNA synthesis associated with recombination,}

TABLE 2

Interspecies (S. typhimurium Hfr3E. coli F) recombination in different recombination and/or

mismatch repair-deficient mutants

E. coli F23S. typhimurium Hfr

Ratio of

Recombination Experiment mutH mutS mutS to

Recipient frequency no. effect effect mutH effects

Arg1/SA977 Hfr

1. mut1rec1 6.831026_61.531026 ₁₁

2. mutH 1.531024_60.431024 _{5 22}

3. mutS 5.031023_62.231023 _{5 735 33}

4. uvrD 3.131025_60.631025 ₃

5. uvrD mutH 1.6310236_0.63₁₀23 _{5 52}

6. uvrD mutS 7.6310236_3.63₁₀23 _{4 245 5}

7. recD 3.1310256_1.33₁₀25 ₅

8. recD mutH 3.731023_61.831023 _{4 119}

9. recD mutS 7.1310236_2.63₁₀23 _{3 229 2}

10. recG 3.1310266_1.53₁₀26 ₆

11. recG mutH 1.4310246_0.43₁₀24 _{3 45}

12. recG mutS 3.331024_62.331024 _{10 106 2}

13. ruvA 6.331027_60.331027 ₃

14. ruvA mutH 1.331025_60.931025 _{7 21}

15. ruvA mutS 5.731024_63.731024 _{3 905 44}

16. lexA1 3.831027_60.331027 ₅

17. lexA1 mutH 4.031026_60.931026 _{7 11}

18. lexA1 mutS 4.2310256_1.03₁₀25 _{4 111 11}

Arg1/SA965 Hfr

19. mut1rec1 3.3310266_1.03₁₀26 ₃

20. mutH 7.9310256_1.13₁₀25 _{4 24}

21. mutS 1.6310236_0.063₁₀23 _{3 485 20}

22. recF 5.731027_60.0631027 ₃

23. recF mutH 5.1310266_2.73₁₀26 _{6 9}

24. recF mutS 2.9310256_0.23₁₀25 _{4 51 6}

Leu1/SU573 Hfr

25. mut1rec1 1.231026_60.331026 ₃

26. mutH 1.331025_60.331025 _{3 11}

27. priA 3.931027_62.531027 ₄

28. priA mutH 8.831027_65.531027 _{4 2}

Conjugational crosses are described inmaterials and methods. The results are the median values of at

least three independent experiments. The analysis is focused on the relative effects of mutH and mutS mutations (see text). Mut1rec1strain is E. coli K-12; AB1157. Conjugation time was 40 min for crosses involving SA977 and 60 min for SA573 and SA965 donors.

quired for editing by MutH depends on PriA activity. tion involving DNA synthesis. The significance of this finding and the observation that the lexA1 and ruvA The effect of RecF on editing by MutH protein is less

pronounced (MutH effect is still ninefold). Perhaps by mutations also decrease interspecies more than intra-species recombination (Table 2;Maticet al. 1995; data coincidence, it is quantitatively closer to the effects of

lexA1 (11-fold). not shown) are discussed below.

The difference between these results and those of a Table 2 shows that a priA null mutation and a recF

mutation inhibit interspecies recombination by z15- previous study on the implication of the recF gene prod-uct in interspecies recombination (Matic et al. 1995) fold in mutH background, while having a twofold and

negligible effect, respectively, on intraspecies recombi- derives from the difference in leakiness of the two recF alleles used. It has been reported that the recF143 substi-nation (data not shown). Thus, PriA and RecF functions

are more limiting for interspecies than intraspecies re- tution mutation used in the previous study, among its other leaky phenotypes tested, exerts an effect in conju-combination in an otherwise wild-type genetic

back-ground. This effect is more pronounced in MutH-defi- gational recombination in recBCsbcBC recipients 10 times weaker than that of the recF349 deletion allele cient bacteria, presumably because of the efficient

TABLE 3 to prevent the dissociation of such unstable recombi-nation initiation events, particularly when MutH is The effects of mutH mutation and of Dam methylase

not active (Table 2; Figure 2e). Recombination-asso-overexpression on interspecies recombination

ciated DNA synthesis has been postulated for some between S. typhimurium Hfr and E. coli F2

systems of homeologous recombination in yeast E. coli F23S. typhimurium Hfr (Porteret al. 1996; Tranet al. 1997).

3. Direct maturation of the recombination process can

Recombination Experiment

be provided by branch migration of the Holliday

Recipient frequency no.

junction away from the invading end into the domain

Arg1/SA977 Hfr _{of diverged parental sequences (Figure 2d). About}

mut1rec1 7.560.431026 ₄

a 10-fold decrease of interspecies recombination by

mutH 1.560.431024 ₅

the ruvA mutation (Table 2, lines 13–15) suggests

mut1pDam 1.260.131024 ₃

that RuvAB provides the branch migration function

mutH pDam 1.661.231024 ₅

(Maticet al. 1995). This activity may be SOS

induc-Leu1/SA534 Hfr

ible (ShurvintonandLloyd1982; see the effect of

mut1rec1 3.961.931027 ₃

the lexA1 mutation in Table 2, lines 16–18) because

mutH 1.160.131025 ₃

mut1pDam 1.360.131025 ₂

interspecies conjugation induces SOS response

mutH pDam 1.260.131025 ₃

(Maticet al. 1995).

4. MutH-independent or early-stage editing of interspe-The mut1rec1strain is E. coli K-12; AB1157. Conjugation

cies recombination corresponds to the dissociation time was 40 min.

of the earliest RecA-catalyzed heteroduplex interme-diate (Figure 2c) because of mismatch formation. This editing requires the key mismatch recognition

A model for the editing of interspecies

recombina-and repair functions, MutS recombina-and MutL, as well as the

tion:These results can be organized within the frame

UvrD (helicase II) function. Helicase II can become of a model involving two mechanisms for editing of

dispensable at very high concentrations of the MutS interspecies recombination in E. coli: one is MutH

inde-and MutL proteins (S. Sˇtambuk and M. Radman, pendent and the other is MutH dependent (Figure 2).

unpublished results). This mechanism was probably These two mechanisms could act in parallel as two

alter-reproduced in vitro in the study by Worth et al. native mechanisms: one is DNA end dependent but

(1994). The effect of the recG mutation on interspe-MutH independent, and the other is dependent on

cies recombination in the mutS mutant (Table 2, MutH, unmethylated GATC sequences, and de novo

lines 10–12) suggests that the RecG protein may be DNA synthesis. However, because of the requirement

recombinogenic only in the mutS mutant (compare of DNA strand transfer for de novo DNA synthesis, we

lines 3 and 12 in Table 2) or that the MutS-mediated, hypothesize that the MutH-independent editing is an

early-stage editing, whereas the MutH-dependent pro-cess is a late-stage editing. The key features of the model

TABLE 4 are as follows:

Interspecies recombination (E. coli Hfr3S. typhimurium

1. The recombination between diverged DNAs is

lim-F2) and the mutH mutation effect in crosses involving ited by the number of minimum efficient processing _{parents with different Dam methylation status} segments (MEPS), i.e., minimal blocks of sequence _{(see text for explanations)}

identity that are sufficient for the efficient

RecA-catalyzed initiation of the DNA strand transfer or S. typhimurium F23E. coli Hfr exchange process (ShenandHuang1986;Vulicet

Recombination Experiment

al. 1997). The outcome of the initiated strand inva- _{Recipient frequency no.} sion process (including its reversibility,Rayssiguier

Met1/PK3 Hfr et al. 1989) will depend on several recombination and

1. mut1rec1 4.5 61.131026 ₂

repair proteins acting on intermediates of genetic

2. dam 2.5 60.331025 ₂

recombination. _{3. mutH 4.7 61.1310}25 ₃

2. Because the sequence divergence between the re- _{4. dam mutH 1.1 60.4310}24 ₃

combining DNAs limits the length and/or stability _Met1_{/PK3dam Hfr}

of the initial heteroduplex intermediate, the inter- 5. mut1rec1 3.8 60.431026 ₂

6. dam 2.7 60.631025 ₂

species recombination becomes more dependent on

7. mutH 5.0 63.631025 ₃

recombination-associated DNA replication than the

8. dam mutH 2.8 62.431024 ₃

isogenic intraspecies recombination. The PriA- and

RecF-dependent de novo initiation of DNA synthesis _{The mut}1_rec1_{strain is S. typhimurium LT2; SL4213.} Conjuga-tion time was 40 min.

Figure 2.—(a–g) A

model for two mechanisms of editing of homologous recombination by the mis-match repair proteins. The MutH-independent (early-stage) editing of homolo-gous recombination during the course of bacterial con-jugation is directly related to the degree and pattern of sequence divergence be-tween the two recombining DNAs. This editing (c–b) occurs before extensive branch migration and/or re-combination-initiated DNA replication. The MutH-de-pendent (late-stage) editing (e to g) occurs after the ini-tiation of DNA synthesis. Both mechanisms of editing by the mismatch repair components require MutS and MutL proteins: the early-stage editing requires specifically UvrD helicase and RecBCD nuclease, and the late-stage editing re-quires specifically the MutH protein (see text for de-tails). Newly synthesized DNA strands are presented by thick lines, and mis-matches are represented by triangular bumps in the du-plex DNA. REC1and REC2 refer to high and low recom-bination frequencies, re-spectively. (111) Overex-pression of MutS, MutL, and Dam proteins.

MutH-independent editing isz10 times less efficient 1994). The RecA protein could use the aborted sin-gle-stranded end to reinitiate the strand invasion in the recG mutant (Table 2, line 12). Curiously, a

functional interaction was postulated between a process that leads eventually to the repeated (and therefore more successful) attempts to initiate recom-RecG homolog of S. pneumoniae and some undefined

mismatch repair function (Martinet al. 1996). bination in the rare regions of sufficient sequence identity (MEPS, see Shen and Huang 1986). In-This (early) stage of editing presumably occurs

be-fore the initiation of DNA synthesis (and/or RuvAB- spired by the results of Zahrtand Maloy (1997), F. W. Stahl suggested to us that the RecBCD nuclease catalyzed branch migration) and does not involve

site irreversible by the breakdown of the dissociated pression (Tables 3) are well accounted for by this model (Figure 2h).

single-stranded end (see below). (Note that unlike events in isogenic recombination, the destruction of

The lack of a coherent effect of Dam methylation of a donor MEPS sequence condemns the RecABCD

parental DNAs on MutH- (and unmethylated GATC-) system to search for the next MEPS within the

dou-dependent editing of recombination (Table 4) leads to ble-stranded Hfr DNA, which, at 16% divergence

the prediction that the functional interaction between from the recipient chromosome, may be many

kilo-the mismatch recognition by MutS/L and kilo-the GATC bases away from the Hfr DNA end). Consequently,

recognition by MutH cannot cross the Holliday junction in a recD null mutant, the repeated strand invasions

(see Figure 2 and imagine a GATC sequence to the left would decrease the global effect of MutS on

recombi-of the Holliday junction). If it could, then the MutH nation as observed (Table 2, lines 1–3 vs. 7–9). In

effect should be at least as efficient in the case of un-this scheme, the RecBCD nuclease is required to

methylated parental GATCs as in the case of newly syn-improve the efficiency of the early-stage editing.

thesized GATCs because the mismatch repair process Therefore, the frequency and stability of the

interme-should necessarily destroy the three-stranded junction diate (c) should increase in the recD mutant, and the

in the former case. This implies that for efficient MutS, impact of the late-stage editing (the MutH effect) is

L, and H activity, the mismatch and the GATC sequence expected to increase, which was found to be the

must be strictly “in cis” on the same strand of a hetero-case (see the next paragraph). The role of RecBCD

duplex DNA. Experiments with structural barriers be-nuclease in early-stage editing is also reflected in the

tween the mismatch and the GATC sequence should observation that the contribution of this mechanism

test this prediction. is not significant in the RecF and RecE

recombina-Comparison with homeologous recombination in

tion pathways (S. SˇtambukandM. Radman,

unpub-other systems:The conspicuous resemblance in the ge-lished results).

netic requirements between the interspecies recombina-5. Late-stage editing is defined operationally as the

tion and the adaptive frameshift mutagenesis in E. coli MutH-dependent editing that appears to act on the

(Foster 1998; Rosenberg et al. 1998) may be more DNA strand that contains a mismatch in the

hetero-than accidental. The key intermediates in the adaptive duplex region derived from the parental sequences

mutagenesis proposed by Rosenberg and colleagues and an unmethylated GATC sequence in the newly

(Harriset al. 1996) are identical to the intermediates synthetized extension of the same strand (Figure 2e).

in Figure 2. Could it be that the privileged sites for The results in Tables 2–4 support this conclusion.

adaptive mutations are created by recombination-associ-Curiously, UvrD (helicase II) function is not required

ated DNA synthesis? Such a process seems to occur in for this editing step. In fact, the increase of

interspe-yeast and involves the REV3 DNA polymerase ( Strath-cies recombination caused by the mutH mutation is

ernet al. 1995;HolbeckandStrathern1997). even higher in the uvrD mutant (52-fold) than in the

In yeast, meiotic interspecies (S. cerevisiae 3S. para-wild-type strain (22-fold; Table 2, lines 4–6). This

doxus) recombination (Hunteret al. 1996) and several difference may result from the higher incidence of

mitotic recombination systems showed effects of null the late-stage intermediates caused by the inefficient

mutations in gene homologs of bacterial mutS and mutL early-stage editing in the uvrD mutant and, hence,

genes (Alaniet al. 1994;Selvaet al. 1995;Dattaet al. more impact of the MutH-dependent editing. [The

1996, 1997) similar to those in bacterial crosses. How-same holds for the recD and recG mutants (Table

ever, in some yeast homeologous recombination systems 2, lines 7–12).] Alternatively, another helicase may

associated with transformation, the effects of mismatch replace the missing helicase II and be even more

repair mutations appear to range from weak to nonexis-efficient in this late-stage editing step. This editing

tent (Mezardet al. 1992;Priebeet al. 1994;Porteret step resembles an incomplete mismatch repair

pro-al. 1996). cess that leads to the separation of the two parental

Gene replacement by homologous recombination be-molecules (Figure 2g) involved in the attempted

re-tween a linear donor DNA fragment and the chromo-combination event that has already initiated DNA

some in the mouse embryonic stem cells is highly sensi-synthesis. We postulate that the unwinding/excision

tive to the natural sequence divergence (0.6%), and tract extends from the unmethylated GATC to the

this effect can be totally accounted for by the activity Holliday junction, thus interrupting the

recombina-involving a mutS homolog gene (MSH2, de Windet al. tion process by separating the two parental DNA

1995). This result suggests that the practice of gene molecules (Figure 2, e–g).

therapy by gene replacement will have to take into ac-6. Coincident branch migration and DNA replication

count the effects of DNA sequence divergence (poly-(Figure 2f) should lead to the productive one-end

morphism) that differentiate donor and recipient DNAs recombination event associated with an unscheduled

and those of mismatch repair systems. Last but not least, replication fork. Equal and nonadditive effects of the

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