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 se- quence 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