Instability of Repetitive DNA Sequences: the Role of Replication in Multiple Mechanisms

Colloquium

Instability of repetitive DNA sequences: The role of replication in multiple mechanisms

Malgorzata Bzymek* and Susan T. Lovett†

Department of Biology and Rosenstiel Basic Medical Sciences Research, Center MS029, Brandeis University, 415 South Street, Waltham, MA 02454-9110 Rearrangements between tandem sequence homologies of various neuromuscular disorders, such as Fragile X, Huntington’s dis- lengths are a major source of genomic change and can be delete- ease, and myotonic muscular dystrophy, are associated with rious to the organism. These rearrangements can result in either expansion of a trinucleotide repeat array (8). Given that local deletion or duplication of genetic material ﬂanked by direct se- rearrangements between dispersed or tandem repetitive sequence repeats. Molecular genetic analysis of repetitive sequence quences contribute significantly to genetic instability in pro- instability in Escherichia coli has provided several clues to the karyotes and eukaryotes, an important question is whether there underlying mechanisms of these rearrangements. We present are underlying common mechanisms for these rearrangements. evidence for three mechanisms of RecA-independent sequence rearrangements: simple replication slippage, sister-chromosome Materials and Methods exchange-associated slippage, and single-strand annealing. We Bacterial Strains and Growth. All strains used are derived from the discuss the constraints of these mechanisms and contrast their E. coli K-12 strain AB1157 [FϪ thi-1 hisG4 ⌬(gpt-proA)62 argE3 properties with RecA-dependent homologous recombination. Rep- thr-1 leuB6 kdgK51 rfbD1 ara-14 lacY1 galK2 xyl-5 mtl-1 tsx-33 lication plays a critical role in the two slipped misalignment supE44 rpsL31 racϪ ␭Ϫ; ref. 9] and carry the additional mutant mechanisms, and difﬁculties in replication appear to trigger rear- alleles indicated. Experimental strains were constructed by P1 rangements via all these mechanisms. virA transduction (10). Details of the constructions are available on request from the authors and will be published elsewhere. LB ␮ ͞ n bacteria, systematic study of repetitive sequence instability medium was supplemented with 100 g ml ampicillin or 15 ␮ ͞ Ihas provided some insights into the molecular mechanisms of g ml tetracycline. repetitive sequence rearrangement. In this paper, we will review the genetic properties of tandem repeat rearrangements in Deletion Assays. Plasmid pMB301 was constructed by ligation of Ј Escherichia coli that are informative about the mechanisms of synthetic oligonucleotides of the sequence 5 GATCTTGGG these processes. Although repetitive sequences can rearrange to AGCTTGTTCT TGAGCATTCA AACTCCTAGA GGAA- Ј either increase or decrease the number of repetitive elements, GAAGAA CGTAGC and 5 GATCGCTACG TTCTTCTTCC the process of deletion of repeated DNA sequences has been TCTAGGAGTT TGAATGCTC AAGAACAAGC TCCCAA more widely characterized and will dominate our discussion. in the BglII site of pSTL57 (11). The appropriate construction Nonetheless, many of the properties of repeat amplification (or was confirmed by DNA sequence analysis. Deletion between the expansion) are similar to those defined for repeat deletion (1). 101-bp direct repeats in tetA on plasmids pSTL57 and pMB301 We will propose and contrast several molecular mechanisms by was assayed as described (11) by determination of the number of which tandem repeats rearrange: homologous recombination, tetracycline-resistant colony-forming units (cfu) in the ampicil- simple slipped misalignment, sister-chromosome slipped mis- lin-resistant population for a total of 8–64 independent isolates. alignment, and single-strand annealing. We present new data in Deletion rates were calculated by the method of the median (12) ϭ ͞ support of a single-strand annealing mechanism. The homolo- by using the following formula: deletion rate M N, where M gous recombination pathways have been well studied in E. coli is the calculated number of deletion events and N is the final r (2) and mediate interactions of repeated DNA in many contexts. average number of Ap cells in the 1-ml cultures. M is solved by This article will focus on the other more genetically elusive interpolation from experimental determination of r0, the median r ϭ ϩ molecular mechanisms. These mechanisms contribute to rear- number of Tc cells, by using the formula r0 M(1.24 lnM). A 95% confidence interval was determined as described (13). rangements between repeats found in direct orientation and COLLOQUIUM constitute the RecA-independent recombination pathways of E. coli. Discussion of RecA-independent recombination can be Linear Transformation Assays. Purified pSTL57 plasmid DNA found subsumed under the term ‘‘illegitimate’’ recombina- (prepared with miniprep kits from Qiagen, Chatsworth, CA) was tion—we favor the more descriptive and specific ‘‘RecA- subjected to digestion by BglII restriction endonuclease, which independent’’ recombination to denote these pathways. cleaves between the two 101-bp direct repeats in this plasmid. Rearrangements between repetitive sequence elements un- The linear fragment was isolated by agarose gel electorophoresis derlie many examples of genomic instability in both prokaryotes and purified (QIAquick gel purification kit from Qiagen). and eukaryotes. A large subset of mutations that inactivate genes Electroporation (ref. 14; Bio-Rad Gene Pulser) was used to are deletion events between two short regions of sequence homology, both in bacteria and in humans (3–5). In addition to This paper results from the National Academy of Sciences colloquium, ‘‘Links Between rearrangements between dispersed repeated DNA sequences, Recombination and Replication: Vital Roles of Recombination,’’ held November 10–12, instability of repeated sequences juxtaposed in tandem is also 2000, in Irvine, CA. observed. Rearrangements causing either addition or deletion of Abbreviations: ssDNA, single-strand DNA; DSB, double-strand break; SCE, sister-chromo- one or more of the sequence repeats can arise. In humans, some exchange. deletion or duplication between repeated DNA sequences con- *Present address: Department of Molecular and Cellular Biology, Harvard University, 7 tributes to human genetic disease, both of nuclear genes and in Divinity Avenue, Cambridge, MA 02138. the mitochondrial genome (4, 6, 7). Several genetically inherited †To whom reprint requests should be addressed. E-mail: [email protected].

www.pnas.org͞cgi͞doi͞10.1073͞pnas.111008398 PNAS ͉ July 17, 2001 ͉ vol. 98 ͉ no. 15 ͉ 8319–8325 Downloaded by guest on September 27, 2021 transform100 ng of cleaved and purified DNA into the appro- exhibit a Tex phenotype (30–32). The function of Uup is not priate bacterial strains. Uncut and purified pBR322 DNA was known but it appears to be a general suppressor of RecA- transformed in parallel as a control for the efficiency of trans- independent rearrangements because RecA-independent tan- formation. After serial dilution in 56͞2 buffer (15), the number dem repeat deletion (unassociated with transposons) is also of transformants was determined by plating on LB medium enhanced in uup mutants (32). Some tex mutants (including containing 100 ␮g͞ml ampicillin. Plating within experiments was those inactivating the dam mutHLS uvrD- dependent mismatch performed in duplicate. repair pathway) appear to be somewhat specific to transposon deletion, by stabilizing a mismatched stem-loop structure Results and Discussion formed by the long inverted repeats of the transposon (32), Homology-Dependent, RecA-Independent Recombination. Deletion which is stimulatory to the excision events. Other tex genes may or duplication of tandem repeat sequences in E. coli shows weak have more general effects on deletion, including ssb [single- or no dependence on RecA DNA pairing and strand exchange strand DNA (ssDNA) binding protein], topA (topoisomerase I), protein (11, 16–19). In contrast, homologous genetic recombi- polA (DNA polymerase I), and recBC (subunits of Exonuclease nation scored by conjugation crosses is highly dependent on the V). Increased persistence of ssDNA during replication (as in ssb RecA, with reductions by greater than five orders of magnitude or polA mutants) may encourage slipped mispairing (described seen in recA mutant strains (20). Although RecA-independent below). Mutations in topoisomerase I may increase the proba- tandem repeat rearrangements can occur between very short bility of secondary structure formation of the inverted repeats by regions of sequence homology, it is nonetheless homology- elevating supercoiling density. Inactivation of the potent driven. The homology dependence of repeat deletion is quite RecBCD exonuclease may encourage rearrangements by stabi- striking (16, 17, 21), with large repeats deleting at very high rates lizing broken DNA molecules (see below). in the population. So, although short sequence homologies suffer The role of special sequences in promoting high-efficiency deletion rarely, in the range of spontaneous point mutations, RecA-independent recombination is largely unexplored. Be- deletion of large repeats approaches the efficiency of homolo- cause many of the models for RecA-independent recombination gous RecA-dependent recombination. RecA-dependent recom- invoke strand displacement during DNA replication, replication bination of tandem repeats, on the other hand, appears to stall sites might be imagined to be hotspots for deletion. In require a threshold of minimal homology, although that minimal polymerase I mutants, CTGG and GTGG sequences are found length may be somewhat context-specific. One study found that to flank sites of spontaneous deletion (33). The Tus͞Ter system for homologies less than about 200 bp in length, there is little or that arrests replication helicase movement evokes nearby RecA- no appreciable contribution of RecA-dependent recombination independent deletion events at short homologies (34). Inverted to the observed deletion events (21). For repeats larger than repeat sequences with the potential for secondary structure about 200 bp in length, deletion can occur by RecA-dependent formation promote deletion at flanking short sequence direct homologous recombination, in addition to the RecA- repeats (29, 35–39). (Deletion associated with inverted repeats independent pathway (21). In our hands, there is no detectable will be discussed specifically in a section below.) RecA- RecA-dependent recombination contributing to deletion of independent deletion of tandem repeats has been primarily 100-bp repeats (11); in contrast, approximately 2͞3 of the studied on ColE1-based plasmids for experimental ease, but has deletion events between 787-bp repeats occurs by the RecA also been observed for tandem repeats on the E. coli chromo- homologous recombination pathway (18). some (18, 22). Chromosome context effects on deletion rates The genetic basis of RecA-independent recombination is ill have been noted with the repeats present on a ColE1-derived defined. RecA-independent recombination is also independent plasmid, an F factor, as a lambda prophage or integrated into of the known recombination functions of E. coli, including lacZ of E. coli (40), but the molecular basis of these effects is RecBCD, RecET, RecF, RecG, RecJ, RecN, RecO, RecQ, elusive. RecR, RuvAB, and RuvC (M.B. and S.T.L., unpublished results; ref. 18). Our screens for transposon insertion mutants decreasing Constraints on High-Efficiency RecA-Independent Recombination. Be- the rate of RecA-independent deletion of tandem repeats have cause a mutation in RecA blocks most other homologous failed to yield candidates, suggesting that if genes are required recombination events, what special features allow tandem re- to mediate RecA-independent rearrangements, they may be peats to rearrange at high frequency in the absence of RecA? essential. A number of genes whose mutation leads to elevated The homology dependence suggests that strand pairing is re- rates of tandem repeat deletion have been defined and include quired for RecA-independent recombination, as it is for RecA- many of the components of the replication apparatus (see refs. dependent recombination. However, high-frequency RecA- 13 and 22 and below). Mutation of the 3Ј single-strand exonu- independent rearrangements always involve interactions clease, Exonuclease I (whose gene is named sbcB or xonA), between homologies that can take place intramolecularly—in elevates deletion of both short and long sequence repeats (23, contrast, recombination between homologies on separate DNA 24). Chromosome topology may also play a role: mutants in the molecules, which must occur intermolecularly, exhibits a strong type I topoisomerase, topoisomerase III (topB), have been dependence on RecA. Moreover, proximity of the repeat se- isolated by their hyperdeletion phenotype (25, 26). The mech- quences clearly plays an essential role in RecA-independent anism by which inactivation of topB potentiates deletion forma- recombination. There is an exponential decrease in deletion rate tion is not understood but could include increased negative as the distance between the homologies increases (11, 21, 41). supercoiling or increased formation of a structural intermediate This contrasts to RecA-dependent recombination of tandem sensitive to topo III unwinding. One potential difficulty in the repeats that shows little proximity effect (21). Such high depen- genetic analysis of RecA-independent recombination is the dence on sequence proximity has supported the idea that many existence of more than one RecA-independent pathway, each of these RecA-independent rearrangements occur in the context with a distinct genetic basis. of the replication fork. The requirement of RecA as a strand- Transposon excision is a special case of deletion at short direct pairing protein may be obviated by the proximity of the inter- repeats, promoted by the large inverted repeats of these ele- acting sequences as well as the single-stranded nature of DNA ments (27, 28). Mutations that elevate the precise and nearly in the fork. In E. coli, the average Okazaki fragment is 1–2 kb in precise excision of the transposon Tn10 or Tn5 at short direct length (42), which might indicate that several kb should be the repeats within or flanking the element (29) have defined the upper limit for the distance between sequences that can undergo so-called tex mutants (27, 28). Mutations in the uup gene also efficient RecA-independent recombination.

8320 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.111008398 Bzymek and Lovett Downloaded by guest on September 27, 2021 Support for a Replicative Mechanism. Several genetic properties of RecA-independent recombination support the idea that these rearrangements occur during the process of chromosomal replication. In E. coli, mutations in many of the components of the DNA PolIII holoenzyme result in elevated levels of tandem repeat rearrangements (13, 22). This includes the polymerase subunit (␣, dnaE), the proofreading exonuclease (␧, dnaQ), components of the clamp-loading complex (␶ and ␥, dnaX; ␹, holC), and the processivity clamp (␤, dnaN). Mutations in the replicative helicase (dnaB) and single-strand binding protein (ssb-113) that are reported to fail to interact correctly with the polymerase (43) also cause a hyperdeletion phenotype. For dnaE, dnaQ, and holC, genetic analysis has shown that the stimulation of tandem repeat rearrangements is largely RecA- independent (ref. 13; C. J. Saveson and S.T.L., unpublished results). These observations are consistent with the notion that Fig. 1. Replication misalignment (‘‘slippage’’) model for genetic rearrange- conditions that stall polymerization or increase the amount of ments. A slipped alignment of the nascent strain with respect to template can generate deletion or expansion of a directly repeated sequence and any ssDNA in the fork increase the probability of tandem sequence intervening DNA. rearrangements. A switch from normal theta replication to ssDNA synthesis greatly enhances RecA-independent ‘‘nearly precise’’ transposon excision, which did not involve transfer of a simple polymerase ‘‘skip’’ accounts for the rearrangement. parental DNA to the recombinants, again supporting a replica- Rather, it seems likely that the polymerase must dissociate from tive mechanism (44). its template, allowing the nascent strand to dissociate and Our study of ‘‘homeologous’’ (slightly divergent) repeat de- translocate to a new pairing position. Resumption of DNA letion also supports the idea that the majority of RecA- synthesis at the mispaired position then accomplishes the dele- independent tandem repeat deletion events occur during or tion or expansion event. Replication slippage in vitro during shortly after DNA replication (45). The mismatch repair system replication of hairpin structures is correlated with the inability of aborts deletion event between repeats with slight sequence various polymerases to mediate strand displacement and there- dissimilarities; there is no effect of mismatch repair on deletion fore their tendency to be blocked by the hairpin (50). In vivo, between perfectly homologous repeats. In the former case, the active displacement of the nascent strand by helicase action may heteroduplex intermediate formed between the strands of the aid the slipped realignment process, although no specific helicase two repeats presents sequence mispairs that elicit mismatch in E. coli has yet been assigned this role. repair excision, leading to destruction of the potential for The replication slippage model explains the homology and deletion. This action of mismatch repair on homeologous repeats proximity dependence of RecA-independent rearrangements requires E. coli’s system to discriminate between nascent and and why most repeat rearrangements are not reciprocal—that is, parental DNA—that is, the Dam methylase that marks the deletions are not formed concurrent with expansions. Its inti- parental strand and MutH endonuclease that cleaves unmeth- mate association with the replication process explains the hyper- ylated DNA to target excision to the nascent strand. Therefore, recombinogenic nature of many DNA replication mutants, for most deletion intermediates must be found in hemimethylated which polymerization may be less processive and ssDNA may DNA, a state that exists only transiently after DNA replication. tend to accumulate. It is likely that many misalignments involve These results are concordant with the replication slippage model Ј Ј (see below) where the deletion arises by misalignment of the the 3 nascent strand end because mutations in the 3 exonucle- nascent DNA strand during replication. ases, ExoI and DnaQ, substantially elevate RecA-independent deletion (13, 23, 24). However, it is also possible that some The presence or absence of the DnaB replicative helicase Ј complex may channel subsequent processing of stalled or slipped misalignments may involve the 5 end of the Okazaki blocked replication forks. RecA-independent deletion of tan- fragments, as has been proposed in eukaryotes to account for the dem repeats does not require reassembly of DnaB onto the fork high frequency of duplication mutations seen in yeast rad27 mutants that fail to process Okazaki 5Ј ends (51). The compa- via the PriA primosomal protein (C. J. Saveson and S.T.L., Ј unpublished results); in fact, priA mutants show hyper-deletion rable Okazaki processing function in E. coli is the 5 exonuclease (13). This may indicate that RecA-independent strand rear- activity of DNA polymerase I (42).

rangements occur with the DnaB complex still positioned on the COLLOQUIUM fork. On the other hand, mutations that impair the DnaB fork Sister-Chromosome Exchange (SCE)-Associated Slipped Misalignment helicase itself stimulate homologous RecA-dependent recombi- Model. The simple slipped misalignment model, despite its nation disproportionately relative to the RecA-independent pleasing features, cannot account for all of the observed RecA- pathways (13). Polymerase dissociation with the fork helicase independent deletion events. A subset of RecA-independent complex intact may preferentially lead to RecA-independent rearrangements, selected by a decreased or increased number of strand rearrangements; dissociation of the helicase complex tandem repeats, are associated with replicon dimerization (1, from the replication fork may facilitate fork regression and 16–18, 26, 52). These dimeric products are not consistent with breakage and subsequent RecA-dependent recombinational simple slippage of the nascent strand on its template. However, repair (46). crossing over between these circular plasmid replicons can generate circular dimers (Fig. 2). Experiments show that RecA- The Replication ‘‘Slippage’’ Model. An attractive model for RecA- independent dimeric products do not result from intermolecular independent recombination is slipped misalignment of the nas- crossing-over, but rather from intramolecular sister-chromo- cent DNA strand during replication (Fig. 1). Originally proposed some interactions (18). In our experiments selecting gain or loss to explain frameshift mutations in nucleotide sequence repeats of a 787-bp tandem repeat, 21% of selected expansion products (47), this model has been invoked for larger repeat rearrange- and 27% of deletion products are dimeric in structure: among ments (48, 49). However, for these longer-range interactions these dimers, 90% of expansion product dimers and 4% of over hundreds or thousands of bases, it is difficult to imagine that deletion product dimers represent reciprocal exchanges (1, 18),

Bzymek and Lovett PNAS ͉ July 17, 2001 ͉ vol. 98 ͉ no. 15 ͉ 8321 Downloaded by guest on September 27, 2021 of its relative single-strandedness. Several studies in both prokaryotes and eukaryotes support the notion that deletions at short sequence homologies associated with inverted repeats do occur preferentially on the lagging strand (56–58). Perfect palindromes can also adopt cruciform structures in duplex DNA. One study correlated the frequency of excision with the cruciform-forming potential of several inverted repeats (37). By what mechanism do palindromic sequences stimulate RecA-independent deletion? For dispersed homologies, formation of secondary structure at the inverted repeat may serve to increase the local proximity of the direct repeats to facilitate misalignment. In addition, secondary structure can block DNA polymerization (59), increasing the probability of replication slippage events. Secondary structures may be recognized and processed by specific enzymes, creating double-strand breaks (DSBs) or single-strand nicks or gaps, which may be deletion- prone substrates.

Two Mechanisms for Palindrome-Stimulated Deletion: SbcCD-Depen- dent and SbcCD-Independent. To study the mechanisms by which palindromic sequences stimulate deletion, we modified an assay for deletion between 101-bp direct repeats in the tetA gene of pBR322 to include a perfect 57-bp palindrome between the direct repeats, creating pMB301. This can be compared with a similar nonpalindromic construct, pSTL57 (11), with perfectly juxtaposed direct 101-bp repeats. Deletion as scored by this Fig. 2. Unequal crossing-over between circular molecules. Unequal crossing- latter construct has been studied extensively in our laboratory over between direct repeats borne on plasmids generates a circular dimer, with the conclusion that deletions arise predominantly by simple with one triplicated and deleted allele. replication slippage with a minor contribution of SCE-associated slippage events (11, 24, 45, 53). In this set of constructs, no increase in the proximity of the direct repeats can be afforded by with concomitant loss and gain of repeats in the two alleles secondary structure formation between the inverted repeats and carried on the dimeric product (Fig. 2). Reciprocality is consid- therefore any stimulation by the palindrome must be via other ered a hallmark of ‘‘break-join,’’ conservative recombination means. The size of the palindrome was chosen to be well below and, again, is not consistent with simple replication slippage. that which gives rise to marked loss (approximately 200 bp; ref. Mutations in the RuvAB complex that mediate branch migration 60), and we observed no defect in copy number or maintenance of Holliday junctions increase the proportion of RecA- of palindrome-containing constructs relative to controls (M.B. independent deletion events that are recovered as reciprocal and S.T.L., unpublished results; ref. 61). crossover dimeric products 13-fold (18), implicating a branched The presence of the palindrome stimulated deletion formation intermediate susceptible to RuvAB action as an intermediate in by two to three orders of magnitude (Table 1). This stimulation RecA-independent SCE events. A slipped misalignment model was independent of RecA and more dramatic at the lower involving mispairing of the two nascent strands of the sister growth temperature. The natural temperature sensitivity may chromosomes (Fig. 3) has been proposed to explain these results reflect more stable pairing to stabilize the secondary structure (1, 18, 53). It has been proposed that these strand exchange at low temperatures. Transformation efficiency of an incompat- events may reflect a normal fork repair reaction, a template ible plasmid into strains with resident palindrome or nonpalin- switch gone awry because of mispairing at tandemly repeated drome plasmids verified that the stimulatory effect of the sequences. The contribution of this pathway to normal repair of palindrome is not due to its relative inefficiency in competing replication blocks has not been established, however, because no with a newly arising deletion plasmid (M.B. and S.T.L., unpub- mutants that block this pathway have yet been isolated. Although lished results; ref. 61). this mechanism shares many features of the simple slipped The most revealing aspect of our genetic analysis was the effect misalignment model, namely strand displacement and mispairing of the sbcD mutation: part but not all of the palindrome- in the context of a blocked replication fork, several mutations stimulation was found to depend on the sbcD gene (Table 1). stimulate this dimer-producing pathway disproportionately, such This differentiates two mechanisms by which palindrome DNA as sbcB (Exonuclease I) and dnaE (the polymerase subunit of stimulates deletion formation, one SbcD-dependent and one polIII) (13, 24). SbcD-independent. Other palindromic constructs show this property; with these, sbcD also dramatically alters the distribu- Secondary Structure Stimulation of RecA-Independent Tandem Re- tion of deletion endpoints within the repeated sequences (61). peat Rearrangements: A Special Case. Many spontaneous deletions The sbcD gene encodes the nuclease component of the SbcCD between short dispersed DNA sequence repeats are found complex of E. coli (62). In vitro, SbcCD possesses ssDNA associated with nearby inverted repeat sequences (36, 54). endonuclease and double-strand DNA (dsDNA) exonuclease Studies of this phenomenon support the idea that inverted activities (63, 64) and can cleave hairpin structures (65). In repeats greatly stimulate genetic rearrangements at short flank- vivo, SbcCD appears to introduce DSBs at palindromic ing direct repeats, presumably by formation of a stem-loop sequences (66, 67). secondary structure (35, 37, 39, 55). Transposon excision (29) represents a well studied example of deletion between short A Single-Strand Annealing Mechanism for RecA-Independent Deletion direct repeats, stimulated by nearby inverted repeats. Formation Associated with Palindromes. These genetic effects lead Secondary structure formation is expected to be more fre- us to propose that the SbcCD nuclease cleaves cruciform or quent on the lagging-strand template during replication because hairpin structures formed by inverted repeat sequences—this

8322 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.111008398 Bzymek and Lovett Downloaded by guest on September 27, 2021 Fig. 3. Model for SCE associated with replication slipped misalignment, yielding deletion (18). (A) A stalled replication fork with direct repeat sequences shown in black. (B) Displacement of nascent strand 3Ј ends. (C) Misalignment of nascent ends at repeat sequences. (D) Processing of crossed strand to yield Holliday-junction structure. The structure can be processed in two alternative ways. If no realignment of parental strands occurs: (E) repair replication. (F) Junction cleavage to form a reciprocal triplication͞deletion dimeric product. If realignment of parental strands occurs: (EЈ) repair replication. (FЈ) Junction cleavage by RuvC or other resolving activity to form a nonreciprocal deletion͞duplication dimeric product. For deletion, this outcome is favored over that shown in F.InruvAB mutants, FЈ is favored, yielding a higher level of reciprocal products. COLLOQUIUM break is then repaired by RecA-independent single-strand annealing at the repeats after resection of the ends (Fig. 4). The Table 1. Palindrome stimulation of deletion formation single-strand annealing mechanism is well documented in eukaryotes (68–70). Deletion rate ϫ 106 In E. coli, this mechanism does not account for many of the (95% conﬁdence interval) properties of simple tandem repeat deletion and so most likely Growth Strain pSTL57 no pMB301 with does not contribute substantially to deletion that is not associ- temperature genotype palindrome palindrome ated with palindromes. Experiments show that expansions are produced as efficiently as deletion and share many of the same 37° recϩ 5.0 (1.1–1.6) 425 (250–480) genetic properties (1). These cannot be via single-strand anneal- recA 4.7 (3.2–8.4) 220 (150–300) ing because, by its nature, single-strand annealing can only recA sbcD 3.8 (2.0–5.4) 40 (12–66) efficiently produce deletion and not expansion rearrangements recA recB 1.7 (0.4–4.8) 24 (7.0–54) (see Fig. 4). The genetic effects on homeologous repeat deletion 30° recA 1.7 (0.6–7.2) 2400 (1500–5200) are also incompatible with deletion mediated by single-strand Strains used were AB1157, JC10287, STL4494, and JC5547. Mutant alleles annealing but, rather, support a simple replication slippage include ⌬(recA-srl)304 (JC10287, STL4494), recA13 recB21 (JC5547), mechanism as the predominant mode of deletion (45). Deletions sbcD300ϻTn10kan (STL4494).

Bzymek and Lovett PNAS ͉ July 17, 2001 ͉ vol. 98 ͉ no. 15 ͉ 8323 Downloaded by guest on September 27, 2021 Fig. 4. Single-strand annealing model for deletion associated with palindromes. Palindromic DNA can adopt cruciform structure. SbcCD initiates cleavage of the cruciform. Resection followed by annealing at the ﬂanking direct repeats generates a deletion.

Fig. 5. Dissolution of homeologous deletion intermediates by mismatch between repeats containing mismatched bases are aborted by the repair. (A) Slipped misalignment model. MutH incision of the unmethylated methyl-directed mismatch repair (MDMR) pathway, dependent strand removes the slipped strand, thereby aborting the deletion event. In the on strand discrimination via Dam methylase and MutH endo- absence of Dam methylation, the ability to target excision to the slipped nuclease (45). For the slipped mispairing model, MutH- deletion strand is lost, resulting in the observed elevation of homeologous dependent incision and targeted excision of the nascent slipped deletion rates (45). (B) Single-strand annealing model. Mismatch excision of either DNA strand destabilizes the intermediate. However, preexisting nicks strand by MDMR (the top strands in Fig. 5) explains the loss of should obviate the need for MutH. There is likewise no reason to explain the potential deletion products. In the absence of methylation for stabilization of the intermediate when Dam methylase is lacking. strand discrimination, excision of the parental template strand should occur, causing fixation of the deletion; deletion rates of mismatched repeats are indeed greatly elevated by loss of the RecBCD, either directly or indirectly, in the generation of the Dam methylase (45). For the single-strand annealing model (Fig. deletion event. Part of this reduction may be due to cellular 5), it is difficult to reconcile destabilization of deletion inter- inviability (74), or plasmid loss in this strain because rates are mediates dependent on both methylation and the MutH endo- reduced 3-fold for the nonpalindromic control plasmid. Muta- nuclease (which cleaves at unmethylated GATC sites). Although tions in recBC also increase rolling-circle replication, causing mismatch excision may destabilize the annealing intermediate plasmid DNA to accumulate in a linear form (75). Because (Fig. 5), mismatch repair excision proteins should be able to cruciform extrusion is facilitated by negative supercoiling, cru- access the DNA from the broken ends, without requiring MutH ciform formation should be diminished in this linear state. incision. Furthermore, there is no good explanation for the The lack of any RecBCD inhibitory effect on palindrome- rescue of deletion products by the absence of methylation. stimulated deletion may be because SbcCD remains bound to the In E. coli single-strand annealing is inefficient, most likely ends of the DSB and protects it from complete degradation by because of the rampant DNA degradation by the RecBCD RecBCD. Potentially dimerizing through the coiled-coil domain enzyme. For example, healing of linear DNA, either produced in vitro or introduced by transformation, at terminal repeats is Ϫ inefficient unless cells are RecBCD (refs. 71 and 72; Table 2). Table 2. DSB repair after transformation of linear pSTL57 tex RecB and RecC mutations are among the mutations that Repair efﬁciency* elevate deletion of transposons from the E. coli chromosome (27, Strain genotype ϫ 104 % deleted (tetϩ)† 28). The plasmids used in our study lack Chi sites to attenuate RecBCD nuclease (73) and so should be especially prone recA 1.5 100 to RecBCD-mediated degradation. Although mutation abolish- recA sbcD 1.1 82 ing RecBCD nuclease elevated by 10-fold the healing of recA recB 11.0 88 transformed broken DNA with terminal direct repeats (Table 2), Strains and genotypes are listed in Table 1. it did not elevate deletion promoted by SbcCD on palindromic *Repair efﬁciency is the number of transformants for the linear pSTL57 resident plasmids (Table 1). If anything, deletion formation may molecule relative to those for uncleaved pBR322. be reduced in recB mutants, both for palindrome and the †Percent of ampicillin-resistant transformants that are tetracycline-resistant, nonpalindrome-containing constructs. This may implicate indicating deletion at terminal repeats.

8324 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.111008398 Bzymek and Lovett Downloaded by guest on September 27, 2021 of the SbcC protein (62), the SbcCD complex may also bind both Summary. Evidence supports the existence of three RecA- broken ends simultaneously, facilitating their annealing inter- independent mechanisms distinguished by their genetic proper- action. A mutation in sbcD did not affect healing of transformed ties that contribute to genetic rearrangements in E. coli: simple linear DNA with terminal repeats (Table 2), suggesting that replication slippage, SCE-associated replication misalignment, SbcCD’s role in palindrome-stimulated deletion must include and single-strand annealing. For rearrangements between large sequence homologies, these mechanisms can be comparable in the initiation of the break. SbcCD-initiated DSBs may be special their efficiency to RecA-dependent homologous recombination. in that they can heal by RecA-independent single-strand anneal- RecA-independent processes contribute exclusively to rear- ing if there are homologies flanking the break, thus resulting in rangements between short sequence homologies but are con- a deletion mutation. The homology and proximity-dependence strained to intramolecular rearrangements over fairly short of this SbcCD-dependent single-strand annealing mechanism in distances. The single-strand annealing pathway, identified for E. E. coli remains to be determined. If the RecA-dependent coli only recently, appears to contribute primarily to deletions recombination pathways are available, SbcCD-initiated DSBs associated with palindromic sequences and depends on the can also be repaired by nonmutagenic recombination with the SbcCD nuclease, presumably to introduce a DSB at the hairpin sister-chromosome (66, 67). structure formed by the inverted repeats, and possibly to protect The SbcCD-independent form of palindrome-stimulated de- the broken ends. Difficulties in DNA replication contribute to sequence rearrangements via all four mechanisms. letion, we believe, represents simple replication slippage promoted by the hairpin’s block to replication. Analysis of deletion This work was supported by the General Medical Institute of the endpoints with respect to replication direction is consistent with National Institutes of Health Grant RO1 GM51753 and by Predoctoral these blocks occurring predominantly on the lagging strand (61). Training Grant T32 GM07122.

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