Mutational analyses of dinucleotide and tetranucleotide microsatellites in Escherichia coli: influence of sequence on expansion mutagenesis

Kristin A. Eckert 0 1 Guang Yan 0 1 0 Hershey, PA 17033, USA 1 The Jake Gittlen Cancer Research Institute, The Pennsylvania State University College of Medicine , PO Box 850 Mutagenesis at [GT/CA]10, [TC/AG]11 and [TTCC/ AAGG]9 microsatellite sequences inserted in the herpes simplex virus thymidine kinase (HSV-tk) gene was analyzed in isogenic mutL+ and mutLEscherichia coli. In both strains, significantly more expansion than deletion mutations were observed at the [TTCC/AAGG]9 motif relative to either dinucleotide motif. As the HSV-tk coding sequence contains an endogenous [G/C]7 mononucleotide repeat and ~1000 bp of unique sequence, we were able to compare mutagenesis among various sequence motifs. We observed that the relative risk of mutation in E.coli is: [TTCC/AAGG]9 > [GT/CA]10 ~ [TC/AG]11 > unique ~ [G/C]7. The mutation frequency varied 1400fold in mutL+ cells between the tetranucleotide motif and the mononucleotide motif, but only 50-fold in mutL- cells. The [G/C]7 sequence was destabilized the greatest and the tetranucleotide motif the least by loss of mismatch repair. These results demonstrate that the quantitative risk of mutation at various microsatellites greatly depends on the DNA sequence composition. We suggest alternative models for the production of expansion mutations during lagging strand replication of the [TTCC/AAGG]9 microsatellite. - Microsatellite sequences of 14 or 5 nt per repeat unit are ubiquitous throughout the human genome (14). These repetitive sequences can be found flanking coding sequences and within introns, as transcribed but untranslated genomic regions (1,5,6). Unfortunately, our knowledge as to the exact number, sequence composition and genomic location of microsatellites is biased by the large proportion of cDNA sequences that constitute the current genomic sequence databases (2,5,6), and a full appreciation of this class of repetitive sequence must await completion of the various genome projects. Nevertheless, evidence exists supporting a role for [GT/CA]n and [TC/AG]n sequences in the regulation of gene expression (79) and in modulating chromatin structure (10). Moreover, within the past decade, a direct involvement of microsatellite sequences in human disease has been demonstrated (11). Microsatellite sequences influence the local geometry of DNA due to the potential for adopting non-B DNA conformations (10). Repeats of alternating purinepyrimidine bases (e.g., GT/ CA) can form Z-DNA, and repeats of polypurine and polypyrimidine tracts (e.g., TC/AG) can form triplex DNA. In addition, particular trinucleotide sequences (e.g., CGG/GCC) have the potential to form stable hairpin structures (12). The effect of non-B DNA forms on DNA metabolism, including replicative, repair and recombination processes, has not been studied rigorously. The ability of long microsatellite sequences that are capable of forming both triplex and hairpin structures to arrest DNA synthesis in vitro (13,14) forms the basis of current models for preferential genetic expansion of these alleles in vivo (15,16). A large base of knowledge exists in several model systems, including Escherichia coli, yeast and human cells, regarding the genetic stability of mono-, di- and trinucleotide microsatellite sequences (1621). The favored mechanism to explain alterations in microsatellite allele size is slipped strand mispairing between repeat units during replicative or repair DNA synthesis (22,23). Consistent with this model, loss of DNA polymerase proofreading activity or post-replication mismatch repair (MMR) greatly enhances the rate of [A]n and [GT/CA]n microsatellite tract alterations (1618,21,24). However, little is known about the genetic factors controlling tetranucleotide sequence stability. In yeast, the mutation rate for a [CAGT/GTCA]n allele was similar to that of a [GT/CA]n allele, and was increased in an MMR-deficient strain (25). In human cell lines, direct measurements of microsatellite alleles have yielded estimated mutation rates for [GATA/CTAT]n sequences that are significantly higher than rates for [GT/CA]n sequences (26), and the mutation rate for an [AAAG/TTTC]n microsatellite is one of the highest measured for microsatellites in human cells (27). Nevertheless, mathematical modeling of mutation rates at various microsatellites in the genome databases has failed to show a significant difference in mutability between di- and tetranucleotide sequences (28,29). In this study, we compared the stability of the di- and tetranucleotide sequences [TC/AG]n and [TTCC/AAGG]n in MMR-proficient and deficient E.coli strains. Our strategy quantitated the mutability of the microsatellite sequences relative to coding sequences within the same genetic target, the herpes simplex virus type 1 thymidine kinase (HSV-tk) gene. We observed a significantly greater incidence of expansion mutations at [TC/AG]n and [TTCC/AAGG]n alleles, relative to a [GT/CA]n allele. The frequency of mutation at the tetranucleotide locus was up to 40-fold higher than the mutation frequencies at both dinucleotide loci, and MMR affected tetranucleotide stability to only a minor extent. MATERIALS AND METHODS Escherichia coli strains Strain FT334 is a derivative of HB101 (30) with the following genotype: tdk, upp, thi1, hsd20, supE44, lacY1, proA2, ara14, galK2, xyl5, mtl1, leuB6, rpsL20, recA13. Strain PP102 is isogenic to strain FT334, except for the following alleles: recA306 srl::Tn10, mutL::Tn5 (P.Prince and R.Monnat, University of Washington, personal communication) Oligonucleotides used to construct the microsatellite sequences were synthesized by Biosynthesis, Inc (Lewisville, TX) or the Macromolecular Core Facility, Penn State College of Medicine (Hershey, PA). All restriction endonucleases were supplied by Gibco BRL Life Technologies (Gaithersburg, MD) and used according to manufacturers instructions. 5-Fluoro-2deoxyuridine (FUdR) and chloramphenicol were purchased from Sigma Chemical Co. (St Louis, MO). Construction of artificial-microsatellite-containing vectors All artificial microsatellite sequences were inserted in-frame between bases 111 and 112 of the HSV-tk gene, in the sequence context [GT (insert) TCTC]. In the unidirectional vectors described, the first sequence listed serves at the template for the leading strand of replication, and the second sequence serves as the template of the lagging strand. Construction of [GT/CA]10 and [TC/AG]11 microsatellitecontaining plasmids has been described (20), and the same method was used to construct the [TTCC/AAGG]3 vector. The [TTCC/AAGG]9 and [TC/AG]18 microsatellite inserts were synthesized by an in vitro DNA polymerase reaction. A 111 base oligonucleotide, corresponding to the HSV-tk sense strand (nucleotides 73147) and containing the microsatellite sequence to be inserted, was primed by hybridization of a 15mer oligonucleotide at a 1:1 molar ratio. This substrate was used as a DNA template for native T7 DNA polymerase in a (...truncated)