Proc. Natd Acad. Sci. USA Vol. 79, pp. 4128-4132, July 1982 Genetics Model for the participation of quasi-palindromic DNA sequences in frameshift mutation (DNA secondary structure/base-substitution mutagenesis/DNA strand switching/cytochrome c frameshifts/DNA repair) LYNN S. RIPLEY Laboratory of Molecular Genetics, National Institute of Environmental Health Science, Research Triangle Park, North Carolina 27709 Communicated by C. Auerbach, March 29, 1982 ABSTRACT A model is described for the templated produc- based (ref. 2; J. E. Owen, D. W. Shulz, A. Taylor, and G. R. tion of frameshift and base-substitution mutations mediated Smith, personal communication). Thus, alternative models are through aberrant DNA structures arising as a consequence of needed to explain these frameshift mutations. This paper de- quasi-palindromic DNA sequences. Two general mechanisms are scribes the general features ofa model for frameshift mutations considered. One evokes the formation and processingofimperfect arising in quasi-palindromic DNA sequences and suggests some DNA secondary structures (hairpins) for the production of mu- alternative mechanisms by which these sequences generate tations. The other evokes a "strand switch" during DNA synthesis mutations. The model successfully predicts the properties of which, in a manner unique to quasi-palindromic sequences, may frameshift mutations in be resolved to produce frameshift or base-substitution mutations, the iso-l-cytochrome c gene ofSaccha- or both. It is the unique combination of symmetrical and asym- romyces cerevisiae, whose properties are inconsistent with the metrical elements of the quasi-palindromic sequence itself that Streisinger model. provides the basis for both models. Through the mechanisms de- scribed, the symmetrical elements permit unusually paired DNA Complementarity within quasi-palindromic sequences substrates, and the asymmetrical elements permit templated in- sertions, deletions, and base substitutions. The model predicts a Palindromic sequences in double-stranded DNA molecules class of mutations simultaneously frameshifts and base substi- have the inherent property ofself-complementarity within each tutions-whose sequences can be predicted from a local quasi-pal- single strand of the DNA. This complementarity permits such indromic sequence. This prediction appears to be met by a sig- sequences to form uniquely paired DNA structures that are im- nificant fraction (more than 15%) of frameshift mutations in the possible in sequences lacking this complementarity; a classical iso-l-cytochrome c gene of Saccharomyces cerevii'ae. example is the formation ofhairpins or cruciform structures. In quasi-palindromic sequences, where complementarity is im- Additions or deletions ofsmall numbers ofDNA bases resulting perfect, the formation of the unusual DNA configurations still in frameshift mutations represent a sizable proportion of spon- may be possible, but the noncomplementarity at imperfections taneous mutations, and their frequency is often increased by in the palindrome provide the potential to generate mutations mutagenic treatments. Studies offrameshift mutational mech- in a templated and, therefore, predictable manner. anisms have focused upon the DNA sequences in which frame- Among the diverse detailed mechanisms by which quasi-pal- shift mutations arise. In many genetic systems, frameshift mu- indromic sequences might be imagined to produce mutations tation has been correlated with DNA sequences comprised of through aberrantly templated DNA synthesis are two general repeated bases (1-4). This observation, first made in the T4 ly- classes that may be distinguished on the basis ofDNA topology. sozyme gene, led to the suggestion by Streisinger et aL that In one class of mechanisms, the aberrant template is provided repeated sequences mediated additions ordeletions by allowing within a single strand of DNA through the formation of DNA local misalignments ofthe complementary strands of DNA (1). secondary structures (hairpin loops), thus providing a locally Such aberrant DNA intermediates, if formed during replica- double-stranded structure; this case will be referred to as DNA tion, recombination, or repair, could then be the precursors of secondary-structure models. In the second class ofmechanisms, frameshift mutations. These repeated DNA sequences may be the aberrant template is provided by the complementary strand the simple reiteration of a single base as observed in the lyso- of DNA. This strand is utilized as a template ifa strand switch zyme and rI genes ofT4 (ref. 5; J. E. Owen, D. W. Shulz, A. occurs during DNA synthesis. Incorporation ofthis aberrantly Taylor, and G. R. Smith, personal communication) but also may templated DNA into the final product can produce templated be more complex repeats. For example, a frameshift hot spot mutations as well; this class will be referred to as strand-switch- in the Escherichia coli lad gene is a sequence ofthree tandem ing models. Strand-switching models may involve, but do not C-T-G-G units (4). Frameshift mutations arisingatthis sitewere necessarily require, the formation ofhairpin loops. found to be the addition or deletion ofone C-T-G-G unit and, thus, were consistent with the prediction of the Streisinger Secondary structure models model. Although the Streisinger model successfully predicts the ge- An example ofa secondary structure that might form in a quasi- netic outcomes of frameshift events in a number of DNA se- palindromic DNA sequence is shown in Fig. 1. Noncomple- quences, it fails to explain a sizable class ofcharacterized frame- mentary elements within the quasi-palindrome arefoundwithin shift mutations that arise within DNA sequences which do not non-hydrogen-bonded regions of the hairpin loop structure. provide the potential misalignments upon which the model is This configuration of the DNA immediately suggests several mechanisms by which the imperfect complementarity might The publication costs ofthis article were defrayed in part by page charge generate mutations. Such mechanisms are described in Fig. 2 payment. This article must therefore be hereby marked "advertise- with a schematic representation ofthe specific secondary struc- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. ture shown in Fig. 1. Regions A and B in both Figs. 1 and 2 4128 Downloaded by guest on September 26, 2021 Genetics: Ripley Proc. Natl. Acad. Sci. USA 79 (1982) 4129 A T T complementary to B provided by the opposite strand of DNA, A G the result (pathway I ofFig. 2) is the deletion ofbases in region G G B that were noncomplementary to sequence A. Alternatively, G *C ifDNA synthesis in region A is templated by sequence B rather A T normal to mutations are C -G than by the complement A, addition A GA.T produced in sequence A (pathway II ofFig. 2). The added bases A A T B in sequence A are complementary to the unpaired bases in se- C G quence B. DNA sequences in which the asymmetry of the pal- C -GCA indrome is due to noncomplementary bases rather than to dif- ferent numbers ofbases, template base-substitution mutations A T this mechanism. C G by The base substitutions produced are com- A T plementary to the formerly noncomplementary bases in the 53 T A quasi-palindrome. Clearly, quasi-palindromes may contain both T C AA GTG G T A A T T CG G T T G A C A C A unequal numbers of bases and noncomplementary bases and, A G T T C A C C A T T A A G C C A A C T G T G as will be seen below, permit the concerted production of 3t A - T frameshift and base-substitution mutations. T - A In addition to providing a template, the secondary structure G - C may also provide a substrate that actually promotes metabolic T * A events ultimately yielding frameshift and base-substitution mu- 6 CC,%T tations. For example, unpaired regions of DNA hairpins might 6 be particularly prone to endonucleolytic attack. Such nicking, G - C followed by exonucleolytic removal ofunpaired bases and then T . AT by DNA synthesis or ligation (or both) templated by the other G C- side ofthe hairpin, can produce mutations as shown in Fig. 2. T A This series of steps is clearly similar to that of excision repair CG actingon ordinary double-stranded DNA. It may be particularly C cC T attractive to consider the potential contribution to mutations in T quasi-palindromic sequences from enzyme systems that remove A A mismatched bases from double-stranded DNA (6). FIG. 1. A quasi-palindromic DNA sequence. This sequence is lo- There is no requirement that secondary-structure models be cated in the rnIB gene of bacteriophage T4 and includes bases 491 limited to repair events. Intermediates ofDNA replication also through 550 as reported by Pribnow et al. (5). The quasi-palindromic can be imagined to participate. For example, if during DNA portion is shown in one of its potential hairpin forms and extends from synthesis a few bases at the primer terminus located in a quasi- base 503 through base 538. The regions A and B in this figure are to- palindromic sequence should dissociate from its normal tem- pologically the same as regions A and B in the more schematic rep- plate and form a short hairpin structure, extension ofthat struc- resentation in Fig. 2. ture through a region ofimperfect palindromic sequence could generate a mutation. Another instance might be the joining of indicate the largely complementary portions of a single strand the Okazaki fragment intermediates ofdiscontinuous DNA syn- ofthe palindromic sequence. However, the A sequence differs thesis. Ifthe formation ofa secondary structure occurred during from the B sequence by two elements ofasymmetry; both ele- templated replacement of an RNA primer in a region of im- ments are extra bases in sequence B. IfDNA synthesis in region perfect complementarity of the palindrome, mutations would B is templated by sequence A rather than by the sequence fully again be expected. Deletions in B A B 5 A B A B 5.