|
Items in cart [0] |
Questions? Call 888-624-8373 |
PAPERBACK Free Resources |
||||||||||||||||
|
||||||||||||||||
The following HTML text is provided to enhance online readability. Many aspects of typography translate only awkwardly to HTML. Please use the page image as the authoritative form to ensure accuracy. ColloquiumInstability of repetitive DNA sequences: The role of replication in multiple mechanismsMalgorzata 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 lengths are a major source of genomic change and can be deleterious to the organism. These rearrangements can result in either deletion or duplication of genetic material flanked by direct sequence repeats. Molecular genetic analysis of repetitive sequence instability in Escherichia coli has provided several clues to the underlying mechanisms of these rearrangements. We present evidence for three mechanisms of RecA-independent sequence rearrangements: simple replication slippage, sister-chromosome exchange-associated slippage, and single-strand annealing. We discuss the constraints of these mechanisms and contrast their properties with RecA-dependent homologous recombination. Replication plays a critical role in the two slipped misalignment mechanisms, and difficulties in replication appear to trigger rearrangements via all these mechanisms. In bacteria, systematic study of repetitive sequence instability has provided some insights into the molecular mechanisms of repetitive sequence rearrangement. In this paper, we will review the genetic properties of tandem repeat rearrangements in Escherichia coli that are informative about the mechanisms of these processes. Although repetitive sequences can rearrange to either increase or decrease the number of repetitive elements, the process of deletion of repeated DNA sequences has been more widely characterized and will dominate our discussion. Nonetheless, many of the properties of repeat amplification (or expansion) are similar to those defined for repeat deletion (1). We will propose and contrast several molecular mechanisms by which tandem repeats rearrange: homologous recombination, simple slipped misalignment, sister-chromosome slipped misalignment, and single-strand annealing. We present new data in support of a single-strand annealing mechanism. The homologous recombination pathways have been well studied in E. coli (2) and mediate interactions of repeated DNA in many contexts. This article will focus on the other more genetically elusive molecular mechanisms. These mechanisms contribute to rearrangements between repeats found in direct orientation and constitute the RecA-independent recombination pathways of E. coli. Discussion of RecA-independent recombination can be found subsumed under the term “illegitimate” recombination—we favor the more descriptive and specific “RecA-independent” recombination to denote these pathways. Rearrangements between repetitive sequence elements underlie many examples of genomic instability in both prokaryotes and eukaryotes. A large subset of mutations that inactivate genes are deletion events between two short regions of sequence homology, both in bacteria and in humans (3–5). In addition to rearrangements between dispersed repeated DNA sequences, instability of repeated sequences juxtaposed in tandem is also observed. Rearrangements causing either addition or deletion of one or more of the sequence repeats can arise. In humans, deletion or duplication between repeated DNA sequences contributes to human genetic disease, both of nuclear genes and in the mitochondrial genome (4, 6, 7). Several genetically inherited neuromuscular disorders, such as Fragile X, Huntington’s disease, and myotonic muscular dystrophy, are associated with expansion of a trinucleotide repeat array (8). Given that local rearrangements between dispersed or tandem repetitive sequences contribute significantly to genetic instability in prokaryotes and eukaryotes, an important question is whether there are underlying common mechanisms for these rearrangements. Materials and MethodsBacterial Strains and Growth. All strains used are derived from the E. coli K-12 strain AB1157 [F− thi-1 hisG4 ∆(gpt-proA)62 argE3 thr-1 leuB6 kdgK51 rfbD1 ara-14 lacY1 galK2 xyl-5 mtl-1 tsx-33 supE44 rpsL31 rac− λ−; ref. 9] and carry the additional mutant alleles indicated. Experimental strains were constructed by P1 virA transduction (10). Details of the constructions are available on request from the authors and will be published elsewhere. LB medium was supplemented with 100 µg/ml ampicillin or 15 µg/ml tetracycline. Deletion Assays. Plasmid pMB301 was constructed by ligation of synthetic oligonucleotides of the sequence 5′ GATCTTGGG AGCTTGTTCT TGAGCATTCA AACTCCTAGA GGAAGAAGAA CGTAGC and 5′ GATCGCTACG TTCTTCTTCC TCTAGGAGTT TGAATGCTC AAGAACAAGC TCCCAA in the BglII site of pSTL57 (11). The appropriate construction was confirmed by DNA sequence analysis. Deletion between the 101-bp direct repeats in tetA on plasmids pSTL57 and pMB301 was assayed as described (11) by determination of the number of tetracycline-resistant colony-forming units (cfu) in the ampicillin-resistant population for a total of 8–64 independent isolates. Deletion rates were calculated by the method of the median (12) by using the following formula: deletion rate=M/N, where M is the calculated number of deletion events and N is the final average number of Apr cells in the 1-ml cultures. M is solved by interpolation from experimental determination of TO, the median number of Tcr cells, by using the formula r0=M(1.24+lnM). A 95% confidence interval was determined as described (13). Linear Transformation Assays. Purified pSTL57 plasmid DNA (prepared with miniprep kits from Qiagen, Chatsworth, CA) was subjected to digestion by BglII restriction endonuclease, which cleaves between the two 101-bp direct repeats in this plasmid. The linear fragment was isolated by agarose gel electorophoresis and purified (QIAquick gel purification kit from Qiagen). Electroporation (ref. 14; Bio-Rad Gene Pulser) was used to This paper results from the National Academy of Sciences colloquium, “Links Between Recombination and Replication: Vital Roles of Recombination,” held November 10–12, 2000, in Irvine, CA. Abbreviations: ssDNA, single-strand DNA; DSB, double-strand break; SCE, sister-chromosome exchange.
|
|
|||||||||||||||
