Proc. Nati. Acad. Sci. USA Vol. 81, pp. 512-516, January 1984 Genetics Structural intermediates of deletion mutagenesis: A role for palindromic DNA (DNA secondary structure/addition and deletion mutations/deletion mutation specfflcity/evolution) BARRY W. GLICKMAN AND LYNN S. RIPLEY Laboratory of Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 Communicated by Matthew Meselson, September 21, 1983 ABSTRACT A model is presented for deletion mutations ic or quasipalindromic DNA sequences participate in dele- whose formation is mediated by palindromic and quasipalin- tion formation through the juxtaposition of deletion end dromic DNA sequences. It proposes that the self-complemen- points. The inherent self-complementarity of palindromic tarity of palindromes allows the formation of DNA secondary DNA sequences permits the formation of cruciform or hair- structures that serve as deletion intermediates. The structures pin structures in nucleic acids, precisely juxtaposing other- juxtapose the end points of the deletion and thus direct dele- wise distant bases (6). Repeated DNA sequences are not re- tion specificity. While misaligned DNA intermediates that ex- quired. plain deletion termini occurring in repeated DNA sequences The termini of the E. coli lacd deletion S86 cannot be jux- have been described, no explanations have been offered for taposed by a misalignment involving repeated sequences. deletion termini occurring in other sequences. The DNA sec- However, the deletion termini are located precisely at the ondary structures whose formation is mediated by palindrom- ends of a quasipalindromic DNA sequence that includes the ic sequences appear to explain many of these. In this paper, entire deletion. The formation ofa DNA hairpin or cruciform secondary-structure intermediates are described for a series of structure in the wild-type DNA sequence places the deletion spontaneous deletions of known sequence in the lad gene of termini immediately adjacent to one another, rather than Escherichia coli. The model is supported by its failure to pre- separated by their normal linear distance of 27 base pairs dict structures that canjuxtapose simulated deletion termini in (Fig. 1A). This structural intermediate would produce the the lacI gene. We have found a strong association between pal- S86 deletion if it served as a substrate for excision or as a indromic sequences and repeated sequences at lacl deletion template for DNA synthesis (Fig. 1B). The latter process termini that suggests the joint participation of repeated and might be mediated by either DNA polymerase or DNA li- palindromic DNA sequences in the formation of some dele- gase. A ligation across such a hairpin stem might also be tions. Sequences of deletions in other organisms also suggest responsible for deletions if the hairpin served as a substrate the participation of palindromic DNA sequences in the forma- for nucleases that removed aberrantly aligned regions from tion of deletions. ordinary double-stranded DNA. Whatever the precise molecular mechanism(s) responsible Local DNA sequences can strongly influence the frequency for the production of deletions from such DNA structures, of mutation (1). The frequent association of deletion termini the prediction of the model is that the deletion termini are with repeated DNA sequences (2-4) suggests that deletion sequence determined and coincide with the end points of the mutations are no exception. The DNA repeats have been palindromic sequences. Secondary structures whose forma- postulated to mediate complementary base pairing between tion is mediated by palindromes precisely coincide with the two misaligned DNA strands. The subsequent fixation of the deletion termini of S86 (Fig. lA) and 556 (Fig. 1C) and thus interstrand misalignment by DNA replication or recombina- offer an explanation for the formation of these deletions. tion generates a deletion or addition mutation. This model for deletion mutagenesis depends upon the presence of re- Quasipalindromes and Repeats at Deletion Termini peated sequences and is analogous to the model for frame- shift mutations proposed by Streisinger et al. (5) for the pro- A previous analysis of the specificity of deletions in the lacI duction offrameshift mutations in locally repeated DNA se- gene demonstrated that DNA repeats were associated with 7 quences. of 12 spontaneous deletions (3). Our analysis of these same Among spontaneous deletion mutations of known se- deletions revealed that, among the 7 deletions associated quence in the Escherichia coli lacI gene (3), however, nearly with repeats, the specific termini of 5 are also predicted by half (5 of 12) have termini in sequences that are not repeated. palindromic sequences. Fig. 2 illustrates DNA hairpins that The origins of these deletions have thus been mysterious. juxtapose the termini of the S74, S23, and S32 deletions. The Our examination of the DNA sequences that are neighbors quasipalindromes responsible for the hairpins, like those in- to deletion termini has revealed the presence of quasipalin- volved in the S86 and S56 deletions, can precisely explain dromic sequences. Misalignments whose formation is medi- the deletion ends. In other words, even were the repeated ated by palindromes are capable ofjuxtaposing deletion ter- sequence absent, the same deletion specificities (termini) are mini, providing explanations for the specific end points of predicted. Also shown in Fig. 2 is deletion S10. The specific many lacI deletions. termini of this deletion are predicted only by repeats. The fact that S86 and S56 can be explained by palindromic Deletion Specificity Directed by Quasipalindromic Sequences sequences in the absence ofrepeats and S10 by DNA repeats in the absence of a palindrome suggests that either palin- In its simplest form, our model proposes that DNA second- dromes or repeats can direct deletion formation. However, ary structures whose formation is potentiated by palindrom- as discussed below, the striking coincidence of both palin- dromic and repeated sequences at 5 of the 12 deletion end can The publication costs of this article were defrayed in part by page charge points suggests the cooperation of both components. As payment. This article must therefore be hereby marked "advertisement" be seen in Fig. 2, the DNA hairpins permitted by the palin- in accordance with 18 U.S.C. §1734 solely to indicate this fact. dromes can be substantially stabilized by interstrand hydro- 512 Downloaded by guest on September 26, 2021 Genetics: Glickman and Ripley Proc. NatL. Acad Sci. USA 81 (1984) 513 A G-A G A peats. Alternatively, it has been suggested that the role of c C G G the palindrome might be to bring the repeats responsible for AG IT, GI this deletion into substantially closer proximity (7). A A G A C * G C .G Deletion Specificity and Complex DNA Secondary Structures , 650 C 690 5-G-C-A-A-T-C-A-A-A-T-C -T-G-G -A--T-G- 3 The models discussed above provide specific explanations for the origins of 9 of the 12 lacI deletions whose sequences B 5- -3 5- have been determined. The termini of the three remaining 3- deletions are associated with repeats of no more than two c A A bases and do not lie precisely at the ends of quasipalindromic 5'- -3' T I sequences. However, both interstrand and intrastrand mis- 3.'....'r5 T-A T-A alignments of a more complex type offer a means by which 'I termini may be juxtaposed and hence could C CC these deletion C G.C- 715 explain their specificity. The basis for these complex mis- T-A T A com- . 3' _A*T alignments resides in the presence of nearby sequences 69021 plementary to the novel joint created by the deletion. At the 5U- A-G-C--C-C-A-A-A3' novel joint, the DNA sequence immediately to the left of the FIG. 1. Production of deletions by palindromic DNA sequences left terminus of the deletion is joined to the sequence imme- at deletion termini. (A) A DNA secondary structure aligning the diately to the right of the right terminus. The nearby se- ends of the lacI deletion S86 by means of a palindromic sequence. In quence that is complementary to this novel joint permits a this and all subsequent figures the numbering of lacI sequences is misalignment thatjuxtaposes the deletion termini. When this from Farabaugh et al. (3) and the deletion termini are indicated by nearby sequence is a repeat of the novel joint, an interstrand arrows. (B) One mechanism for deletion formation; the DNA sec- misalignment mediates the structure; when the sequence is ondary structure forms within a gapped DNA molecule and this in- related to the novel joint, an intrastrand mis- trastrand misalignment is fixed by DNA synthesis across the stem of palindromically the hairpin, generating the deletion. (C) A DNA secondary structure alignment mediates the structure. aligning the ends of the lad deletion S56 by means of a palindromic The lacI deletions S24 and S42 may arise from structures sequence. that involve interstrand misalignments whose formation is mediated by repeats of the sequences at their novel joints. gen bonding due to the presence of repeats. Alternatively, The S24 ends are juxtaposed by a sequence 53 base pairs the interstrand misalignments permitted by repeated se- away, which is a repeat of 7 bases at the novel joint (3 to the quences can be viewed as being stabilized by intrastrand hy- left and 4 to the right of the deletion) (Fig. 3). Similarly, the drogen bonding due to the palindromes. In the case of S10, in S42 termini are juxtaposed by a sequence 25 base pairs re- which repeated sequences can define the deletion termini, moved by an interstrand misalignment permitting the pairing there is substantial potential for hydrogen bonding within the of 9 out of 10 bases at the novel joint (Fig. 3). deleted sequence. Here, then, intrastrand hydrogen bonding A complex structure that specifies the termini of the S120 might contribute to the stability of the structural intermedi- deletion (Fig.