Copyright 0 1997 by the Genetics Society of America
Enhanced Deletion Formation by Aberrant DNA Replication in Escherichia coli
Catherine J. Saveson and Susan T. Lovett
Department of Biology and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254-9110 Manuscript received September 26, 1996 Accepted for publication February 21, 1997
ABSTRACT Repeated genes and sequences are prone to genetic rearrangements including deletions. We have investigated deletion formation in Eschm'chia coli strains mutantfor various replication functions. Deletion was selected between 787 base pair tandem repeats carried either on a ColElderived plasmid or on the E. coli chromosome. Only mutations in functions associated withDNA Polymerase I11 elevated deletion rates in our assays. Especially large increases were observed in strains mutant in dnaQ the E editing subunit of Pol 111, and dnuB, the replication fork helicase. Mutations in several other functions also altered deletion formation: the a polymerase (dna,the y clamp loader complex (holC, dnaX), and the p clamp (dnaN) subunits of Pol I11 and the primosomal proteins, dnaCandpui. Aberrant replication stimulated deletions through several pathways. Whereas the elevation in dnaB strains was mostly recA- and Zeddependent, that in dnaQstrainswas mostly recA- and Zed-independent. Deletion productanalysis suggested that slipped mispairing, producing monomeric replicon products,may be preferentially in- creased in a dnaQ mutant and sister-strand exchange, producing dimeric replicon products, may be elevated in dnaE mutants. We conclude that aberrant Polymerase I11 replication can stimulate deletion events through several mechanismsof deletion andvia both recA-dependent andindependent pathways.
ANDEMLY repeated DNA sequences, common in The RecA-independent mechanism can mediate dele- T the genomes of many organisms, are vulnerable tion between homologies of several nucleotides, al- to deletions and amplifications. Such rearrangements though the efficiency of deletion is improved dramati- may occur between repeated genes, kilobases in length, cally with increased homology (ALBERTINI et al. 1982; or between short segments, of as little as a few nucleo- DIANOVet al. 1991; WINet al. 1991; PIERCEet al. 1991; tides in length. These tandem repeat rearrangements BI and LIU 1994). are common sources of genetic mutation and human DNA replication may play a role in bothmechanisms genetic disease (MEUTH 1989; KRAWCZAK and COOPER of deletionformation. The events that may trigger 1991; Hu and WORTON1992; NELSON1993). RecA-dependent homologous recombination between Genetic analysis of repeatedsequence rearrange- tandem repeats arenot known butcould include a ments in Escherichia coli has revealed that multiple mech- blocked replication fork. Our genetic studies impli- anisms contribute to theprocess. In particular, we have cated the RecF, RecO and RecR homologous recombi- previously investigated the genetic dependenceof spon- nation proteins of E. coli in the RecAdependent dele- taneousdeletion events between 787 base pair (bp) tion pathway LOVE^ et d. 1993; M. Bzymek and S. T. repeated sequences within the tetA gene of E. coli (Fig- Lovett, unpublished results). These functions are also ure 1) (LOVETTet al. 1993). The majority of recovered required for recombinational repair ofDNA lesions deletions occur by a homologous recombination path- that block replication in the cell (TSENGet al. 1994). way requiring the RecA strand transfer protein. How- Therefore, some tandem rearrangements could result ever, a substantial proportion of these events (30%) from recombinational gap-filling reactions in a blocked occur independently of RecA. replication fork as depicted in Figure 2. Unequal cross- Both RecA-independent and-dependent deletion ing-over between sister chromosomes or recombination formation occurs between homologoustarget se- between the two repeats on the same chromosome dur- quences. However, there is a minimal homology re- ing the course of this repair may lead to deletion of quirement for the Red-dependent pathway and no one of the repeated segments. Alternatively, as shown such requirement for the Red-independent pathway. in Figure 3, a stalled replication fork may be broken; The RecA-dependent mechanism does not appear to recombination of the broken chromosome with its sis- function on homologies less than -200 bp in length ter to restore thefork could result in deletion of tandem (DIANOVet al. 1991; WINet al. 1991; BI and LIU 1994). repeats (ASAI et al. 1994; KUZMINOV1995a,b). DNA replication is considered to be central to the Corresponding author: Susan T. Lovett, Rosenstiel Basic Medical Sci- ences Research Center MSO29, Brandeis University, Waltham, MA RecA-independent mechanisms that lead to genetic re- 0225491 10. E-mail: [email protected] arrangements. Experimental evidence supports a repli-
Genetics 146: 457-470 (June, 1997) 458 C. J. Saveson and S. T. Lovett fetA+ :.:.:.:.:.:.;.:.:..:.:.:.:.:.:.:.:.: :.:.:.:.:.:.:.:..:+:+:.:.:.:
tetAdu EmRV Nrul :.:.:.:.:.:.:.: ...... :::::::::I
FIGLIRE1.-Diagram of the ktA locus and the 787-bp dupli- cation used in these experiments. Duplication of the segment from the EcoRV to the NmI sites of the gene disrupts the tdA gene. Deletion of one copy of the tandem repeat restores the IdA' gene structure and can be selected by tetracycline- resistance. cative mechanism for the RecA-independent deletion of both short (TRINH and SINDEN1991) and long (LO- VETT and FESCHENKO1996) tandem repeats. Exposure C. Branch migration J/ of singlestrand DNA during the replication process may provide an opportunity for tandem sequence ho- mologies to interact. A simple slipmispairing model (Figure 4A) proposes that slipped realignment between the nascent strand and its template leads to deletions or amplifications (STREISINCERet al. 1967; ALBERTINI et al. 1982). Additional models propose that deletionsmay arise by strand misalignment within a replication fork D. Resynthesis J. *rn??%?*?A?Y*?* ..,. .,,,,,!Rx*~:?~w$$~??x
A. deletion products from selected strains. We present evi- dence that aberrantor incomplete replication via DNA polymerase I11 stimulates deletion at tandem repeats via several mechanisms.
J, MATERIALSAND METHODS B. Bacterial strains, media and antibiotics: Strains were grown on LB media: 1% Bacto-tryptone, 0.5% yeast extract, 0.5% sodium chloride and 0.005% thymine, and, for plates, 1.5% agar (WIl.LETTS et dl. 1969). Growth temperatures are indi- cated in the text. Minimal complete media consisted of 56/ 2 salts (WILLETTSet al. 1969),0.2% glucose, 1 pg/ml thiamine, JI 50 pg/ml each of required amino acids and, for plates, 2% C. Degradation and recombination agar. LCG media for preparation of P1 lysates and transduc- with sister chromosome dqradatQn tions consisted of LB media supplemented with 1% glucose and 2 mM calcium chloride, and, for plates, 1% agar. A 0.7% agar concentrationwas used for LB and LCG top agar. Media were supplemented with antibiotics: kanamycin (Km) at 60 pg/ml, tetracycline (Tc)at 8-15 pg/ml, chloramphenicol (Cm) at 15 pg/ml and ampicillin (Ap) at 100 pg/ml (for plasmid resistance genes) and 30 pg/ml (for chromosomal genes). J. Isogenic strains used for deletion assays were constructed by P1 transduction and aredescribed in Table 1. These strains include derivatives of AB1 157 for plasmid deletion assays (A) and STL695 for chromosomaldeletion assays (B). P1 uirA phage lysates and transduction were performed as described (MILLER1992). For many strains, a TnlQkan element linked to the mutation of interest was used for selection in strain FIGURE3.-Replication fork collapse and repair(Kuzminov constructions. In thesecases, control strains were constructed 1995b). (A) Inhibition of polymerization stalls the replication carrying only the TnIOkun element to ensure thatthis marker fork. (B) Nuclease attack of single-strand DNA breaks the did not contributeto the deletionphenotype. Deletion assays fork. (C) Thebroken end invades its sister chromosome. Dur- confirmed that deletion rates in these control strains were ing this step unequal recombination can occur at a repeated within 50% of the wild-type value (data not shown). Mutants sequence (dark hatching). (D) Thefork is reestablished with in dnuQ were assayed for mutator phenotype on rifampin a deletion on one chromosome. plates (at 100 pg/ml). Deletion assays: Deletion formation was measured using association is mediated by interactions with the dnaB plasmid pSTL55 (LOVE= et al. 1993). Thisplasmid is a deriva- tive of pBR322, conferring ampicillin resistance, and contains protein, also the replication fork helicase (TOUGUet al. a tandem repeat within the tetA gene (Figure 1). The dupli- 1994). The primase, like the P clamp, is required at cated region spans 787 bp between the EcoRV and NruI sites the start of each Okazaki fragment, placing a potential of tetA. Plasmid DNA was introduced into strains via electro- greater burden on these functions for lagging strand poration (DOWERet ul. 1988).Chromosomal deletion fre- synthesis. Other proteins that are required for primo- quencies were measured in strains carrying an insertion of bla and tetAdup787 from pSTL55 on the E. coli chromosome some assembly and may also be componentsof the pri- at lacZ (LOVE= et al. 1993). Deletion of one tandem repeat mosome include the piA, @‘B, pzC, dnaC and dnaT results in recovery of a functional tetA gene and confers resis- gene products (ARAI and KORNBERG1981). An alterna- tance to tetracycline. tive primosome assembly occurs at the E. coli origin of Strains containing either thechromosomal or plasmid con- replication, using the dnd protein in addition to dnaB struct were grown at their permissive temperatures on LB plates containing ampicillin. Individual colonies were then and dnaC (FUNNELLet al. 1987; MASN et al. 1990). picked whole, and grown in LB+Ap broth for 2 hr. The cul- In this work, we have investigated whether aberrant tures were diluted in 56/2 buffer and the numberof Ap‘ and replication promotes deletions at repeated sequences. Tc‘cells in the population was determined by plating cell We constructed a series of isogenic strains containing dilutions on LB+Ap and LB+Ap+Tc media. Because of poor mutations that impairselected components of the repli- growth in rich medium, polA and pnA mutant strains were grown on minimal-complete agar plates and in minimal-liquid cation machinery. Because there are three DNA poly- media with the appropriate antibiotics. Deletion assays were merases in E. coli, we have tested the genetic depen- performed for 210 independent cultures in parallel with the dence of deletion on each polymerase gene as well as wild-type control strain. Deletion rates were calculated by the other functions associated with DNA replication. E. coli method of the median (Lw and COULSON1949) for the en- mutant strains were surveyed for effects on deletion tire set of assays using the formula: deletion rate = M/N, where M is the calculated number of deletion events and N formation using both plasmid and chromosomal dele- is the final average number of Ap‘ cells in the 1-ml cultures. tion assays. To gain insight into themechanisms of dele- Mis solved by interpolation from experimental determination tion during aberrant replication, we also analyzed the of ro, the median number of Tc‘ cells determined among the 460 C. J. Saveson and S. T. Lovett
A. Simple slippedmispairing B. Sister-strandslipped mispairing
f Nascent strand wk::~s7~::;*;;~x...... ~.:.~.~.:.:.~.:.:~...."...... :.:.:.:. '.' 1II ...... ????>% ...... ".::.:.A...... E?%dR???%?.,.!?...... :* ...... :.:.:.. Sisterstrands anneal + Slippedalignment ..,>,>..:.:.:.>:<.:.:.: ...... W? VS .....
w.s-<:- ...... *I + Monomer deletion product + Dimer deletion product FIGURE4.-Slipped realignment mechanisms for deletion formation. (A) Simple slippage. Displacement and realignment of a nascent strand with its template can delete one copy of a tandem repeat. On a circular chromosome, such deletion products should be replicon monomers. (B) Sister-strand slipped mispairing. Nascent strands are displaced and mispair with each other, causing a deletion to be formed. [Although we have illustrated leading strand synthesis impeded relative to the lagging strand, the endsof the nascent strands may be created by nucleolytic cleavage rather than representing the point atwhich DNA synthesis has stopped. SeeLOVEIT et al. (1993) for furtherdiscussion of this model.] With pairing of the parental strands, a recombinational intermediate is produced. Resolution or replication through the Holliday junction intermediate causes sisterchromosome exchange. Crossing-over between circular sister chromosomesproduces a dimeric deletion product. Both mechanisms are presumed to operate independently of RecA. cultures, by the formula r,, = M( 1.24 + In M). The observed AB1157 strain background that carry various replication numbers of Tc' cells for a given data set were ranked and a mutations (Table 1). Deletion of tandem direct repeats 95% confidence limit for the median was established using Table A-25a of DIXON andMASSEV (1969); this was converted was measured using both a plasmid-based and chrome to a 95% confidence interval for deletion rates as suggested somal assay. Plasmid pSTL55 (LOVEIT et aZ. 1993), con- by Wierdl et al. (1996) using the formula above. taining a 787-bp internal tandem repeat disrupting the Analysis of plasmid deletion products Ted' plasmid dele- tea gene on plasmid pBR322, was introduced into each tion products were examined by electrophoresis to determine monomerand dimer product distribution. Thesedeleted strain. Precise deletion of one repeat restores a functional products were readily distinguishable from each other and tea gene and tetracyline resistance to the cell. The chre from the parental plasmid. Each product examined was iso- mosomal assay used the identical 787-bp ,!&A duplication lated froman independentculture. Dimeric plasmid DNA was construct integrated into the chromosomal lac gene (Lo- never detected from nondeleted Apresistant (Tc-sensitive) VEIT et al. 1993). In the case of pnA and pol4 mutants, colonies, confirming that dimerization occurred concurrent with deletion formation. Plasmid DNAwas prepared from deletion rates could be measured only for the chrome each culture by alkaline lysis (SAMBROOKet al. 1989) or by somal construct because of poor plasmid maintenance. phenolchloroform extraction and subjected to agarose gel Deletion rates were calculated from the number of cells electrophoresis. Phenolchloroform DNA extraction was per- in the population that acquired tetracycline resistance (as formed as follows: single colonies were randomly selectedand described in MATERIAIS AND METHODS). suspended in 25 pl of TE (10 mM Tris-HCI, pH 8.0, 1 mM EDTA). For slow-growing strains, individual colonies were Growth conditionsfor the deletion assaysvaried streakedto LB+Tc and incubatedovernight, producing a among the strains tested. Some mutants we tested were larger streak, which was then suspended in 25 p1 of TE. This viable whereas others were conditional for growth. suspension was extracted with 25 pl of phenolchloroform- Whenever possible, null mutant strains were examined isoamyl alcohol (25:24:1 by volume), mixed vigorously for 1 at a 37" growth temperature (Table 2). The mutant min and cleared by microcentrifugation for 2min. The entire supernatant was then subjected to agarosegel electrophoresis. in dnaQ49 is viable at all temperatures but exhibits a temperaturesensitive mutator phenotype; these strains RESULTS were grown and assayed at their restrictive temperature Effects of replicationmutations on deletion forma- of 37". Because ofpoor growth of poMA mutants (JOYCE tion: We constructed a series of isogenic strains in the andGRINDLEY 1984) and priA mutants (MASAI et al. HyperdeletionMutants in Replication 46 1
1994) on rich medium, these strains were grown and Other mutants affecting primosome assembly and assayed on minimal medium at 37” (Table 2B). Strains the replication fork were also assayed. Elevateddeletion with temperature-sensitive lethal phenotypes (Table 2, rates were seen for a temperature-sensitive mutant of C and D) were measured at their permissive tempera- the dnaB helicaseand primosome mutants in dnaC and ture for growth, either 25 or 30”. In each case, we show dnaT, assayed at their permissive temperatures. These absolute deletion rates for the mutant strain and the strains showed a substantial increase in the plasmid de- rate relative to the wild-type control strain under the letion formation as compared to wild-type strains: 20- same growth conditions (Table 2). For the wild-type fold for dnaB107, 11-fold for dnaCl and 15-fold for the strains, the various growth conditions had little effect double dnaC2 dnaTl2 mutants. The dnaBmutantexhib- on the efficiency of deletion formation. ited a large effect in the chromosomal assay with a 130- We found that many replication mutants exhibited fold stimulation of deletion rate. Unfortunately, the higher deletionrates in both plasmid and chromosomal chromosomal deletion assay could not be used for dnaC deletion assays (Table 2). In particular, some mutants and dnaT mutants due totheir extremely poor growth. with defects in DNA polymerase I11 core enzyme exhib- Null mutants in the primosome helicase gene PriA ited high deletion rates. This includes a sevenfold eleva- showed a 10-fold increase in the chromosomal deletion tion of plasmiddeletion rates seen for the temperature- assay and could not be measured in the plasmid assay sensitive dnaE486 (poZC486) mutant, which is defective because of plasmid instability. in the a polymerase subunit itself. However, a minor We conclude from these studies that aberrant DNA twofold effect on chromosomal deletion rates was seen. replication via polymerase I11 often results in high rates Deletion assays ofstrains carrying the antimutatorallele of deletion between tandem repeated sequences. DNA ofdnaE, dnaE925, were normal. Mutants carrying polymerases I and I1 are not required for deletion for- dnaQ49, affecting the 3’ to 5‘ editing exonuclease E mation in E. coli. Recovery of deletions on the chromo- subunit, exhibited dramatic increases in deletion for- some is impaired in dnaNand dnaxmutants with defec- mation for bothassays, with ninefold and 100-fold stim- tive processivity clamp and clamp loader complex. ulation of plasmid and chromosomal deletion rates, re- Is elevated deletion formation in dm mutants red- spectively.Likewise, another allele ofdnuQ, mutD5, dependent or -independent? Both recAdependent and stimulated deletion rates threefold in the plasmid assay -independent pathways contribute to deletion forma- and 600-fold in the chromosomal assay. Nullmutants in tion between repeats of the 787-bpsize assayed. For hoE, the third 8 subunit of the polymerase core enzyme wild-type strains, approximately 70% of the observed complex, were unaffected for deletion formation in ei- deletion is recA-dependent and 30% is recA-independent ther assay. In contrast tothese mutations affecting DNA (LOVETI et al. 1993). The recA-dependent pathway also polymerase 111, deletions of the genes for polymerase I uses the recombination genes, recF, recR and rec0 and (poZA) and polymerase I1 (poZB) had no influence on may reflect a normal recombinational pathway known deletion rates. as the RecF pathway (reviewed in SMITH1989; CLARK Mutants in the P-processivity “clamp” associated with and SANDLER1994). The genetic basis of the recA-inde- polymerase 111, dnaN, gave seemingly contradictory re- pendent pathway is unknown but has been proposed sults in the two assays: 14fold higher deletion rates in to involve a replication-slippage (or realignment) the plasmid assay and eightfold lowered deletion rates mechanism. in the chromosomal assayas compared to wild-type We chose three mutants with elevated deletion rates strains. This latter result is of special interest because (dnaE486, dnaQ49and dnuBlO7) to determine whether it implicates replication as an essential component of the increased deletion formation was due to recA-depen- deletion formationand suggests that when the polymer- dent or -independent processes (Table 3). Plasmid dele- ase clamp is impaired, formationor recovery ofdeletion tion rates for dnaE486 mutants were independent of events on the E, coli chromosome is reduced. The dis- recA, with an 11-fold stimulation in recA- derivatives crepancy between the two assays will be addressed in compared to sevenfold in recA+ derivatives. (The chro- the DISCUSSION below. mosomal assay had shown no significant elevation and Other mutants affecting the “clamp loader” (the y so was not tested).Assays of the dna449 mutant strains complex of the DNA polymerase I11 holoenzyme) were showed only a minor effect of recA (twofold elevation analyzed. Deletion formation in temperature-sensitive in recA- dnaQ49mutants vs. ninefold elevation in recA+ mutants of the dnaXgene, which encodes both T and dnaQ49 mutants for the plasmid assay; 52-fold stimula- 6 subunits of polymerase 111, was at normallevels for the tion in recA- dnaQ49 us. 98-fold in recA+ dnaQ49deriva- plasmid assay but 30-fold reduced in the chromosomal tives forthe chromosomal assay). In contrast to the assay, reminiscent of the dnaN results above. A null above, assaysof the dnaB107 derivatives showed that mutant in hoZC, encoding the x subunit of the y com- most of the large increase seen in the chromosomal plex, exhibited a fivefold elevated deletion rate in the assay was dependent on the recA gene: deletions rates plasmid assay and a 16-fold elevated rate in the chromo- were 130-foldelevated in recAf but only twofoldelevated somal deletion assay. in recA- derivatives ofdnaB107strains. Plasmid deletion TABLE 1
Escherichia coli strains
Strain Relevant genotype Derivation A. Strains used in plasmid deletion assays AB1157" rec+ BACHMANN(1996) DM49 lexA3 A. J. CLARK,MOUNT et al. (1972) JC10287 A(srlR-recA)304 CSONKAand CIARK(1979) RM4193 holE202: : cat R. MAURER, SIATERet al. (1994) STL1270 dnaBl07(ts) malE::TnlOkan Km' Ts transductant P1 STLll52 X AB1157 STL1316 dnaB107(ts) malE: :TnlOkan A(srlR-recA)304 W Cys' transductant P1 JC10287 X STL1277 STLl336 ApolBl: : spc str ESCARCELLERet al. (1994) STL1818 dnuE486(ts) zae-3095: : Tn 1Okan Km' Ts transductant P1 STL1155 X AB1157 STL1845 dnaN159(ts) rid-3162::TnIOkan Km' Ts transductant P1 STL1205 X AB1157 STL1861 dnaQ49( ts) zae-3095:: Tn 1Okan Km' Mut transductant P1 STL1368 X AB1157 STL1866 priA2: : kan Km' transductant P1 PN103 X AB1157 STL1880 dnaX2016(ts) zba-3101: : Tn lOkan Km' Ts transductant P1 STL1157 X AB1157 STL1909 dnaT12(ts) dnaC2(ts) zji-3188::TnlOkan Km' Ts transductant P1 STL1147 X AB1157 STL2107 holCl02:: cat Cm' transductant P1 RM4848 X AB1157 STLZ 134 SUlAll Pyr+ Tc' transductantRDK1397 P1 X STL774 STLZ 182 sulAI I led 71 : : Tn5 Km' transductant P1 RDKl397 X STL2181 STL2234 dnaE486(ts) zae-3095 : : Tn 1Okan A(srlR-recA)304 uv' Cys+ transductant P1 JC10287 X STL2174 STL2314 dnaQ49( ts) zae-3095:: Tn I Okan A(srlR-recA)304 UV Cys+ transductant P1 JC10287 X STL2267 STL2545 dnaCl (ts)zj-3188: : Tn 1 Okan Km' Ts transductant P1 STL2519 X AB1157 STL'2674 dnaE925 zae: : TnlOd-cam Cm' Km' transductant P1 NR11258 X STL1819 STL2702 priA2: : kan suolll Km' transductant P1 STL1866 X STL'2134 STL2706 dnaE925 zae: : TnlOd-cam A(srlR-recA)304 uv' Cys+ transductant P1 JC10287 X STL2704 STL3000 mutD5 Pro' Mut transductant P1 RDK1276 X AB1157
B. Strains used in chromosomal deletion assays STL695 lad:: bla' tetAdup787 LOVETTet al. (1993) STL753 A(srlR-recA)304 lacZ: : bla' tetAdup787 LOVETTet al. (1993) STLl324 dnaBIO7(ts) maE:: Tn 1Okan lacZ:: bla+ tetAdup787 Km' Ts transductant P1 STL1152 X STL695 STL1353 ApolBl : : spc str lacZ: : bla+ tetAdup787 Ap' transductant P1 STL695 X STL1336 STL1885 dn&486( ts) zae-3095 : : Tn 1Okan lacZ: : bla' tetAdup 787 Km' Ts transductant P1 STL1155 X STL695 STL1887 dnaNI59(ts) zid-3162:: TnlOkan lacZ::bla+ tetAdup787 Km' Ts transductant P1 STL1205 X STL695 STL1902 dnaX2016(ts) zba-3101::TnlOkan lacZ::bla+ tetAdup787 Km' Ts transductant P1 STL1157 X STL695 STL1957 dnaQ49(ts) zae-3095:: Tn 1Okan lacZ: : bh' tetAdup787 Km' Mut transductant P1 STL1368 X STL695 STL2077 holE202:: cat lacZ::bla' tetAdup787 Cm' transductant P1 RM4193 X STL695 STL2082 priA2:: kan lacZ::bla' tetAdup787 Km' transductant P1 PN103 X STL695 STLZ 109 holClO2: : cat LacZ: : bla+ tetAdup787 Cm' transductant P1 RM4848 X STL695 STL2125 lexA3 lad:: bla' tetAdup787 Ap' transductant P1 STL695 X DM49 STL2 129 dnaBlO7(ts) malE: :TnlOkan led3 lacZ::bla' tetAdup787 Km' Ts transductant P1 STL1152 X STL2125 STL2131 dnaQ49(ts) led3 zae-3095::TnlOkan lacZ::bla+ tetAdup787 Km' Mut transductant P1 STL1368 X STL2125 STL2 149 ApoL4:: kan lacZ: : bla' tetAdup787 Km' transductant P1 SClOl X STL695 STL2181 sulAll lacZ: : bla+ tetAdup787 Ap' transductant P1 STL695 X STL2134 STL2 186 dnaQ49(ts) zae-3095:: TnlOkan A(srlR-recA)304 lacZ: : bla+ uv' Cys+ transductant P1 JC10287 X STL2133 tetAdup 787 STL2332 suolll lexA71::Tnj lacZ::bla+tetAdup787 Ap' transductant P1 STL695 X STL2182 STL2385 dnaBlO7(ts) malE:: TnlOkan A(srlR-recA)304 lacZ:: bla+ UV Cys' transductant P1 JC10287 X STL2128 tetAdup787 STL3022 mutD5 lacZ: : bla+ tetAdup787 Pro+ Mut transductant P1 RDK1276 X STL695
C. Intermediate strains derived from AB1157
RM4848 holCI02:: cat recA430 sr1::TnlO zjg-2086::kan R. MAURER STL672 cysC95 : :Tn IO Tc' transductant P1 CAG12173 X AB1157 STL700 cysC95 : : Tn IO lacZ: : bla+ letAdup 787 Tc' transductant P1 CAG12173 X STL695 STL774 pyrD34 zcc-282: : Tn IO Tc' Pyr- transductant P1 DC305 X AB1157 STL1277 dnaB107(ts) malE::TnlOkan cysC95::TnlO Tc' transductant P1 CAG12173 X STL1270 STL2128 dnaB107(ts) malE::TnIOkan cysC95::TnlO lacZ::bla+ Km' Ts transductant P1 STLll52 X STL700 tetAdup787 STL2133 dnaQ49( ts) zae-3095: : Tn IOkan cysC95: : Tn 10lacZ: : bla' Km' Mut transductant P1 STL1368 X STL700 tetAdub787 STLZ 174 dn&48i(ts) zae-3095::TnlOkan cysC95::TnlO Tc' Ts transductant P1 CAG12173 X STL1818 Hyperdeletion in Replication Mutants 463
TABLE 1 Continued
Strain Relevant genotype Derivation
STL2267 dna449( ts) zae3095: :Tn 1Okan cysC95:: Tn IO Km' Mut transductant P1 STL1368 X STL672 STL2704 dnaE925 zae:; Tn 1Od-cam cysC95: : Tn 10 Tc' transductant P1 CAG12173 X STL2674
D. Other strains AX727" dnaX20I 6(ts) = dnaZ2016 B. BACHMANN, FILIPet al. (1974) CAG12107' zba-3101:: Tn 1Okan SINGERet al. (1989) CAG12119' malE: :Tn 1Okan SINGERet al. (1989) CAG12173' cysC95:: Tn 10 SINGERet al. (1989) CAG18558' zid-3162: : Tn 1Okan SINGERet al. (1989) CAG18580' zae-3095 : : Tn 1Okan SINGERet al. (1989) CAG18619' y'j-3188: : Tn 1 Okan SINGERet al. (1989) DC305" zcc-282 : Tn 10 pyrD34 B. BACHMANN E107' dnaB107(ts) B. BACHMANN,WECHSLER and GROSS(1971) E48d dnaE486( ts) = polC486(ts) B. BACHMANN,WECHSLER and GROSS(1971) HC 194" dnaN159(ts) B. BACHMANN,SAKAKIBARA and MIZUKAMI(1980) KH1366" dna449(ts) B. BACHMANN,HORIUCHI et al. (1981) MElO1' ApolBl : : spc str ESCARCELLERet al. (1994) NR1125W dnaE925 zae: : Tn I Od-cam R. SCXAAPER,FIJALKOWSKA and SCHAAPER(1995) PC1 dnaCl B. BACHMANN,CARL (1970) PC2k dnaTl2(ts) dnaC2( ts) B. BACHMANN,CARL (1970) PN103' priA2: : kan K. MARIANS, NURSEet al. (1991) RDKl276" mum5 R. KOLODNER RDK1397" lexA 71 ; : Tn5 sul4l I R. KOLODNER SC101" ApolA: : kan ApolBl: : spc str M. GOODMAN STLl 147k dnaT12(ts)dnaC2(ts) zjj"jr188::TnlOkan Km' Ts transductant P1 CAG18619 X PC2 STLl152' dnaBlO7( ts) malE: : Tn 1Okan Km' Ts transductant P1 CAG12119 X E107 STL1155/ dnaE486( ts) = polC486(ts) zae-3095:: Tn I Okan Km' Ts transductant P1 CAG18580 X E486 STL1157" dnaX2016( ts) zba-3101:: Tn 1Okan Km' Ts transductant P1 CAG12107 X AX727 STL1205" dnaN159( ts) zid-3162: : Tn I Okan Km' Ts transductant P1 CAG18558 X HC194 STL1368" dnaQ49( ts) zae-3095:: Tn 1Okan Km' Mut transductant P1 CAG18580 X KH1366 STL251gk dnaCl (ts) zjj-3188: : Tn I Okan Km' Ts transductant P1 CAG18619 X PC1 " Genotype of AB1 157, STL695 and their derived strains, unless otherwise indicated, includes F- X- hisG4 argE2 leuB6 A(gpt- proA)62 thr-1 thi-1 rpsL31 galK2 lacy1 ara-14 xyl-5 mtl-1 kdgK5l supE44 tsx-33 @Dl mtl-1 rac-. I' I' Genotype of AX727 and its derivative includes F- thi lac rpsL rpsL. 'All CAG strains are derived from MG1655 and their genotype includes F- X-. "Genotype of DC305 includes F- galK2 mal41 xyl-7 mtl-2 rpsL118. 'Genotype of E107 and derivative includes F- thr-1 leuBGJhwl21 supE44 X- @Dl thyA6 rspL67 thildeoC1. /Genotype of E486 and its derivative includes F- thr-1 leuB6 tonA2l lacy1 supE44 X- @Dl thyA6 rpsL67 thil deoC1 met89. "Genotype of HC194 and its derivative includes Hfr tow422 ompF627 reoll thyA148 metBl TT. Genotype of KH1366 and its derivative includes Hfr metD88 Alac-6 tsx-7 X- srl-8 reUl spoT1 metB1. 'Genotype of ME102 includes F- X- tonA2l thrl leuB6 lacy1 supE44 @Dl thil. I Genotype of NR11258 includes F- aru thi A(pro-lac) mutL::Tn5. Genotype of PC1, PC2 and their derivatives includes F- leuB6 X- thyA47 qsL153 deoC3. ' Genotype of PN103 includes HfrC X-. Genotype of RDK1276 includes lacZ trpA metE. Genotype of RDKl397 includes F- leuB6 thr-1 pomet xyl mtl gal ara. "Genotype of SClOl includes F+ A(ga1-bio) thi-1 rel4l spoT1.
rates were also somewhat recA-dependent, with 20-fold survival after productionof replication-blocking lesions elevation inrecA' dnaB107strains and 4.5-fold elevation in DNA. Some replication mutant strains such as dnuQ in recA- dnaB107 strains. We conclude that both recA- and PriA exhibit constitutive expression of the SOS re- dependent and -independent deletion formation can sponse (NURSEet al. 1991; SLATERet al.1994) and it be induced by aberrant DNA replicationand that indi- is unknown what the consequence of this may be on vidual replication mutants differ in the mechanism by observed tandem deletion rates. which deletion formation is stimulated. Induction of the SOS response was neither necessary Effect of the SOS responseon deletion forma- nor sufficient to induce deletion formation (Table 4). tion: The SOS response in E. coli is the coordinated Two lexA mutant strains, one of which (Ind-) blocks induction ofa set of genes whose functionis to promote induction of the SOS response and the other (De0 464 C. J. Saveson and S. T. Lovett
TABLE 2 Deletion rates in red' background
Plasmid assay Chromosomal assay Temperature Deletion rate Relative rateDeletion Relative Genotype (deg) (~10~) rate n (X lo6) rate n A. + 37 1.5 (0.85-2.0) =I 16 5.1 (3.4-7.9) =I 15 dnaE925 37 1.0 (0.82-1.9) 0.66 12 ND dnaQ49 (ts) 37 14 (7.8-19) 9.3 16 500 (200-810) 98 16 dnaQ (mutD5) 3.9 37 (2.8-6.5) 2.6 12 3100 (2200-11,000) 610 22 holCl02 : : kan 37 8.1 (6.3-14) 5.4 21 80 (53- 110) 16 16 holE202:: cat 1.8 37 (1.3-2.7) 1.2 19 3.4 (3.0-6.0) 0.67 16 ApolBl 1.8 37 (1.3-2.0) 1.2 24 5.3 (3.1-7.8) 1.o 16 B. + 37 ND 6.7 (4.7-12) =1 32 ApolA 37 ND 3.4 (2.3-4.5) 0.51 30 priA2: : kan 37 ND 66 (55-97) 22 9.9 c. + 30 1.1 (0.59-1.6) 4.8 =1 24 (3.8-7.2) =1 16 dnaBl07 (ts) 30 22 (18-36) 20 16 600 (470-750) 130 16 dnaE486 (ts) 30 8.0 (7.3-12) 7.3 25 9.8 (5.6-18) 2.0 16 dnaN159 (ts) 30 15 (13-21) 14 11 0.64 (0.60-1.1) 0.13 24 dnaX2016 (ts) 30 0.84 (0.39-1.0) 0.76 16 0.17 (0.11-0.62) 0.035 11 D. + 25 0.95 (0.74-1.2) =I 37 ND dnaCl 25 10 (4.3-19) 11 24 ND dnaTl2 dnaC2 (ts) 25 14 (9.8-17) 15 17 ND All strains are derivatives of AB1157 and are described in Table 1. The strains in A and B were assayed at 37" and the strains in C and D were assayed at permissive temperatures of 30 and 25", respectively. The strains in A, C and D were grown on LB medium, while the strains in B were grown on minimal complete medium. All mutant strains were assayed in parallel with the wild-type control strains AB1157 or STL695. Rates were calculated by the method of the median (LEA andCOULSON 1949) and represent an estimation of the number of deletion events that occurred in the population, calculated from the observed median number of Tc' colonies for the number of independent cultures (n),as described in the MATERIALS AND METHODS. Numbers in parentheses correspond to 95% confidence values for the measured rates. Relative rates were determined by comparison to the control strain assayed under the same growth conditions. ND, not determined. causes constitutive expression of SOSregulated genes, However, the SOS response may augment the deletion showed wild-type levels of deletion formation in both rate in this strain somewhat, which accounts for the the plasmid or chromosomal assays. Furthermore, the twofold decrease in rates seen in both recA and ZexA large increase in deletion formation promoted by E mu- Ind- dnuQmutant derivatives relativeto rec+ kx+ dnuQ tations does not requirethe SOS response with the strains. In contrast to dnaQ a substantial proportion of double mutant dnaQ49 kxA3 (Ind-) showing a 47-fold increased deletion formation in dnuBlO7 strains was increase of deletion formation in chromosomal assays. ZexA-dependent. Deletion rates on the chromosome
TABLE 3 Deletion rates in red derivatives
~~~ ~ Plasmid assay Chromosomal assay Temperature rateDeletion Relative Deletion rate Relative GenotvDe (deg) (~10~) rate n (X106) rate n A. A recA304 0.69 37 (0.38-1.3) =1 16 5.2 (2.7-5.7) =1 16 ArecA304 dnaQ49 (ts) 37 1.2 (0.48-2.3) 1.7 19 270 (60-610) 52 12 B. ArecA304 30 0.51 (0.28-0.56) =1 23 5.3 (2.9-7.6) =1 16 A recA304 dnaB107ArecA304 (ts) 30 2.3 (1.1-5.7) 4.5 19 9.5 (6.3-1.7) 1.8 16 ArecA304 dnaE486 (ts) 30 5.4 (2.3-7.8) 11 23 ND All strains are derivatives of AB1157 and are described in Table 1. The strains in A were assayed at 37" and the strains in B were assayed at a permissive temperature of 30°, in parallel with ArecA304 control strains JC10287 or STL753. Rates were calculated for the numberof independent cultures (n)as described in MATERIALSAND METHODS.Values in parentheses correspond to the 95% confidence limits for the rates. Relative rates were determined by comparison to the control strain assayed under the same growth conditions. ND, not determined. Hyperdeletion in Replication Mutants 465
TABLE 4 Deletion rates for kxA derivatives
Plasmid assay assayChromosomal TemperatureDeletion rateRelative rateDeletion Relative Genotype (deg) (~10') rate n (X 106) rate n Zed3 (Ind-) 37 ND 4.3 (2.2-8.6) 0.84 16 Zed3 (Ind-) dnaQ49 37 ND 240 (91- 280) 47 12 Zed3 (Ind-) dnuBl07 (ts) 30 ND 140 (47-350) 29 15 SUlAll 1.1 37 (0.76-1.6) 0.73 20 9.2 (5.7-14) 1.8 12 sulAll Zed71 (De0 1.7 37 (1.1-2.8) 1.1 20 12 (8.3-22) 2.4 16 All strains are derivatives of AB1157 and are described in Table 1. The strains were assayed in parallel with the wild-type control strainsAI31157 or STL695. Rates were calculated for the numberof independent cultures(n) as described in MATERIAIS AND METHODS. Values in parentheses correspond to the 95% confidence limits for the rates. Relative rates were determined by comparison to the control strain assayed under the same growth conditions. ND, not determined. were29-fold elevated overwild-type strains for Zed3 are found in the monomeric form.The transformation dnaBlO7 compared to 130-fold for lex+ dnaBlO7 deriva- efficiency ofmonomer and dimerforms into these mu- tives. Mutations in the DnaB fork helicase and primo- tants, with and without a coresident monomerplasmid some protein therefore promote deletions, in part, via (data not shown), confirmed that the skewed product a mechanism requiring induction of the SOS response. distribution is not merely due to a changein the estab- Analysis of plasmid deletion products reflects two lishment or maintenance ofvarious plasmid forms. types of Red-independent deletionformation: There Rather, these results suggest that polymerase mutation are several mechanisms that may give rise to deletion dnaE486 increases the likelihood of a dimer-producing events between tandem duplications. Unequal sister- deletion event, such as sister-chromosome exchange chromosome exchange can lead to deletion formation; (see Figure 4). The editing subunit mutation dnaQ49, such events fuse circular replicons and can be detected on the other hand, increases the likelihood of mono- in the plasmid assay by the formation of dimeric plas- mer-producing mechanism such as slipped mispairing mid deletion products. Other mechanisms of deletion on thesame strand (as shown in Figure 4). Theantimu- formation, such as slipped mispairing, are expected to tator polymerase mutation dnaE925 and helicase muta- produce only monomeric products (see Figure 4). tion dnaBl07have no effect on the distribution of dele- Examination of the plasmid deletion products for tion products. Because dnaB107 caused a 4.5-fold their monomeric or dimeric structure can therefore be stimulation of recA-independent deletion, both dimer- informative about the particular deletion mechanism and monomer-producing pathways must be equally en- in play.However, product analysisin recA+ strains is hanced. confounded because of the occurrence of recombina- tion between plasmids before and after the deletion DISCUSSION event. For this reason, we have examined products in the recA strain background, where there is no detectable We have demonstrated that mutations in many repli- plasmid recombination (and plasmid dimerization) in cation genes affect deletion events at tandem repeats. the absence of thedeletion event. Among deletion For most of the mutant strains examined, we observed products in recA strains, 40% were dimeric plasmid increased deletion of repeats both carried on a productsthat represent intramolecular recA-indepen- multicopy plasmid and on theE. colichromosome. How- dent sister-chromosome exchange. The remaining60% ever, for dnaQ and dnaB mutantsthere were much of products were monomeric, formed presumably by larger increases in chromosomal as compared to plas- slipped mispairing ( LOVETTet al. 1993). mid deletion rates. This discrepancy may be due to To determine whether mutations in the replication several reasons. (1) The differences between plasmid machinery affect one of these mechanisms specifically, and chromosomal data may reflect a real difference in we analyzed the plasmid deletion products of selected the way that replication occurs on the plasmid us. the replication mutants (Table 5). Among dnaE486 ArecA chromosome. Both the chromosome and the plasmid mutants assayed at their permissive temperature, where depend on Pol I11 for normal replication although the an 11-fold stimulation of recA-independent deletion is structure of the replication fork could differ in some observed, proportionately more products are found in way [there are differences in the genetic requirements the dimeric form.Conversely, in dnaQ49 ArecA strains, for initiation and primosome assembly (KORNBERGand where recA-independent deletion events are stimulated BAKER1992)l. (2) The chromosome may be subject to twofold relative to dnaQ+ ArecA strains, more products different recombination or repair reactions than occur 466 C. J. SavesonLovett and S. T.
TABLE 5 complete replication and loss of the potential deletion Deletion product analysis for dnuB, dnaE and dnaQ mutants product. Of E. coli's three known DNA polymerases, onlyfunc- Growth tions associatedwith Polymerase 111, responsible for temperature Dimer chromosomal replication, affected deletion at tandem Genotype products repeats in our experiments. This includes mutations in A. A recA304 37 32/82 (39) the a polymerase (dnaEgene) and E exonuclease ArecA304 dnaQ49 (ts) 37 15/93 (16) (dnaQ) subunits of the coreenzyme aswell as mutations ArecA304 dnaE925 37 32/82 (39) in subunits of the y complex (dnaX, hoZC) and the ,B B. ArecA304 30 48/140 (34) clamp (dnaN). Mutants in priA, dnaB, dnaC and dnaT ArecA304 dnaBlO7 (ts) 30 39/107 (36) with defects in the primosome or primosome assembly ArecA304 dnaE486 (ts) 30 (62)54/87 also exhibited high deletion rates. Another mutant of All strains are derivatives of AB1157 and are described in Polymerase 111, the antimutator dnaE (FIJALKOWSKAand Table 1. The strains in A weregrown at 37" and in B at SCHAAPER1995) did not show altered deletion rates. 30". Dimer product ratios represent the number of deletion Deletions of genes encoding Polymerases I and I1 had products that were dimeric plasmids (sister-chromosome ex- no effect on deletion rates in otherwise wild-type strains. change events) out of the total number of plasmid deletion This result does not, however, exclude the possibility products examined for each strain, with the percentage in that these polymerases also contribute in a minor way parentheses. The alternative product was monomeric plas- mid, equivalent topBR322. x' analyses showed that thedistri- to deletion formation. The ssb-113 mutation of single- butions of products in strains dnaE486 and dnaQ49 were sig- strand DNA binding protein had beenpreviously shown nificantly different from control strain ArecA304. to elevate deletion formation fourfold in the same plas- mid assay asused here (LOVETTet al. 1993).This mutant on plasmids.For example,the plasmidlacks se- protein has normal DNA binding properties and is be- x lieved to be defectivein protein-protein interactions quences thatcould stimulate recombinational reactions (CHASEet al. 1984) with components of the replication on the chromosome. (3) Differences observed may re- complex. flect relative recovery of products. For multicopy plas- We believe that deletion formation is stimulated in mids, deletion intermediates may be selectively lost. In- these mutants by aberrant DNA replication rather than deed, plasmid loss may be magnified in mutants that other indirect effects. The phenotype of elevated dele- impair replication, causing reduced recovery of plasmid tion frequency is not correlatedwith slow growth. Dele- deletion products. Determination of plasmid copynum- tion rates in wild-type werefound to be largely indepen- ber (data not shown) suggested that dnaB and dnaC dent of growth medium and temperature. Whereas dnaT mutants, in particular, have two- to fivefold low- some slow-growingreplication mutants showed elevated ered levels of plasmid DNA, which could lead to an deletion rates (dnaB), others showed essentially wild- underestimate of plasmid deletion rates in these strains. type deletion rates despite poor growth (poZA). In addi- The other replication mutants tested here were not tion, our results suggest that induction of the SOS re- significantly affected in this analysis (data not shown). sponse does not itself stimulate deletion formation nor In two cases, the dnaN and dnaX mutant strains, we is it required for theelevated rate seen in some hyperde- observed seemingly contradictory results: no effect or letion mutants, as in the case of the dnaQ mutant. Al- an increase in deletion events for repeats on the plas- though this suggests that aberrant replication in a vari- mid but a large decrease in deletions of the same re- ety of replication mutant strains can lead to enhanced peats on the chromosome. These mutations affecting deletion formation at tandem repeatedsequences, this the processivity clamp may truly affect tandem repeat does not necessarily mean that deletion events in wild- deletion in opposite manners on a plasmid us. a chro- type strains occur strictly during the process of replica- mosome replicon. An alternative explanation, which we tion. Recently, we have presented genetic evidence that prefer, is that, in these mutant strains, chromosomal most deletions formed between 101-bp tandem repeats deletion products may be less likely to be recovered. occur during, or shortly after, DNA replication because For instance, aberrant or incomplete replication may they can be subject to correction dependent on hemi- initiate deletion formation on both the plasmid and methylation (LOVETTand FESCHENKO1996). Moreover, the chromosome. However, on the chromosome, the the reduced recovery of chromosomal deletion prod- deletion intermediate may fail to be processed into a ucts in dnaNand dnaxmutantsalso impliesthat replica- viable deletion product. Because the plasmid presents tion is an essential component of the deletion process. a shorter replication template, it is possible for ineffi- More detailed analysisof several selected mutants cient Pol I11 replication or uncoupled leading and lag- (dnaB, dnuEand dna4) suggested that aberrantreplica- ging strand replication to be completed or replaced by tion can stimulate deletion through different pathways. repair replication via Pol I or 11. On the much larger The hyperdeletion phenotypes for polymerase mutants chromosome, the same situation may lead to failure to in dnaQ and dnaE were largely independent of recA, Hyperdeletion in Replication Mutants 467 whereas that for dnaR helicase mutant was primarily Replication gap rPcAdependent. Also, a functional SOS response was “:::z&Rw>>>>>. A,.,. :.:*, %... ,..%..x.< necessary to see the full stimulatory effect of dnaRmuta- tions on deletion formation, in contrast to dnuQ. The Displacement of nascent strand during replication 4 k??:::::::: ::;:::: .:.:.:.:.:.:.:.~.x.:.:.:.:.: = *e :C*S.X.h >x+x.:.:.:.x.:.:.:.:.> FIGURE 6"Prevention of deletion by the exo- nuclease activity of e (dm@ J protein. Simple slipped re- Slipped realignment alignment may be initiated by the displacement of the nascent strand, which sub sequently mispairswith its templatestrand. Exo- nuclease degradation of the displaced single strand or of the mispaired strand by the 3' exonuclease activity 1 1 of DNA polymerase subunit E will abort the deletion event. Deletion on nascent strand ciation of the polymerase could free the nascent DNA late that it is the loss of the exonuclease activity that is strands and allow their translocation across the fork or responsible for the dnaQeffects, E has also been shown on their template strands to produce slipped mispair- to modulate a and the processivity of the polymerase ing. Mutations in the a subunit of the polymerase may (STUDWELLand O'DONNELI.1990) and this property stimulate Red-independent recombination via this may influence the deletionprocess, as described above. mechanism. Our observations that mutations in the replication Other mutants in the p clamp and clamploading/ machinery may greatly stimulate genetic rearrange- recycling machinery y complex may stimulate deletion ments may have implications for human genetic dis- formation by decreasing processivity of the polymerase. ease. Rearrangements between tandem repeated se- We would expect mutations in the clamp or mutations quences or genes are a source of genetic mutation in to the polymerase that hinder its interactions with the humans (MEUTH 1989; KRAWC~AKand COOPER1991; clamp, to inhibit the processivity of the enzyme com- HU and WORTON1992; NEISON 1993). It is likely that plex, thereby increasing the incidence of deletions. As aberrant replication in humans, as in other organisms, with the primosome, one might expect defects in the can also promote deletionevents. Mutations in the anal- clamp loader to affect lagging-strand synthesis more ogous replication functions of human cells to those we than leading-strand synthesis,slowing replication or have investigated here may well predispose certain indi- leaving single-stranded gaps. However, in some cases, viduals for more frequent genetic rearrangements and deletions on the chromosome may not be recoverable genetic disease. because of failure to reinitiate and complete replica- We thank PAOIA DRAPKINand ~.ERGI.UCKMAN forpreliminary tion. work on this project and VIADIMIR FESFIENKOfor construction of the Replication mutations may also stimulate deletion by poL4 mutant strain. We are indebted to the following individuals for allowing slipped mispairing intermediates to persist. We providing strains: B. BACHMANNof the P;. coli Genetic Stock Center, observed that both the dnaQ49 and mutD5 mutation A. J. CIARK,M. GOODMAN,C. GROSS,R. KOI.ODNF,R,K. MARIANS,R. increased deletion events. At least in the case of dnuQ49, MAURERand R. SCIIAAPER.We thank the anonymous reviewer for suggesting thestatistical method for setting95% confidence limits for this increase wasvia a recA-independent mechanism. rate determinations. This work was supported by U.S. Public Health This may be via simple slipped mispairing, as we ob- Service grants R01 GM-51753 to S.T.L. and T32 GM-07122 to C:J.S. served proportionately more monomer deletion prod- ucts in dnaQ49mutants. Figure 6 illustrates that, during LITERATURE CITED normal replication, thenascent strand may be displaced from its template. The E subunit of Pol I11 may degrade AIBERTINI, A. M., M. HOFER,M. P. Chos and J. H. MII.I.ER,1982 On the formation of spontaneous deletion: the importance of this displaced single strand from its 3' end andthereby short sequence homologiesin the generation of large deletions. prevent slipped pairingevents from occurring. Alterna- Cell 29 319-328. tively, E may degrade the 3' end of the nascent strain ARAI, K., andA. KORNRERG, 1981 Uniqueprimed start ofphage 4x174 DNAreplication and mobility of theprimosome in a in the slipped alignment with its template and abort directionopposite chain synthesis. Proc. Natl. Acad. Sci. 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