Rapid, stabilizing palindrome rearrangements in somatic cells by the center-break mechanism

Abstract

DNA palindromes are associated with rearrangement in a variety of organisms. A unique opportunity to examine the impact of a long palindrome in mammals is afforded by the Line 78 strain of mice. Previously it was found that the transgene in Line 78 is likely to be palindromic and that the symmetry of the transgene was responsible for a high level of germ line instability. Here we prove that Line 78 mice harbor a true 15.4-kb palindrome, and through the establishment of cell lines from Line 78 mice we have shown that the palindrome rearranges at the impressive rate of about 0.5% per population doubling. The rearrangements observed to arise from rapid palindrome modification are consistent with a center-break mechanism where double-strand breaks, created through hairpin nicking of an extruded cruciform, are imprecisely rejoined, thus introducing deletions at the palindrome center. Significantly, palindrome rearrangements in somatic tissue culture cells almost completely mirrored the structures generated in vivo in the mouse germ line. The close correspondence between germ line and somatic events indicates the possibility that center-break modification of palindromes is an important mechanism for preventing mutation in both contexts. Permanent cell lines carrying a verified palindrome provide an essential tool for future mechanistic analyses into the consequences of palindromy in the mammalian genome.

FIG. 1.

The center-break mechanism of palindrome rearrangement. A cruciform structure is initiated by local underwinding of the DNA. Each extruded hairpin structure is nicked on one strand near the tip. The two nicks comprise a double-strand break at the symmetry center of the palindrome, which becomes reconnected by NHEJ. Overall the process disrupts the perfectly palindromic sequence by introducing central alterations.

FIG. 2.

(A) Two copies of the original injected fragment are arranged tail-to-tail in Line 78 (1). (P) indicates that the PstI-generated 5′ overhangs of the fragment had lacked the terminal nucleotide of the PstI recognition site. Rectangles represent lacZ repeats; the two inner copies are truncated. The bar indicates the 3.4-kb BamHI fragment used to probe Southern blots. The dotted line indicates the region of hybridization of this probe within the palindrome. (B) Scale map of the integrated palindrome in Line 78 showing the major diagnostic digests used in the present study. Sizes of probe-positive fragments only are given. Left and right sides are defined according to EcoRI digestion. Wavy lines represent the chromosomal sequences. (C) Location of PCR primers relative to landmark restriction sites (details for each primer are given in Materials and Methods). The chromosomal flanking sequences on the left side are patterned in order to distinguish them from right-side sequences in panels C and D. The primer sequences are given in Materials and Methods; only some of the primers are labeled with their names for orientation. Transgene-specific primers are shown in gray and are mapped as they occur within the two identical palindrome arms. Chromosome-specific primers are in black; left- and right-side primers are not identical. (D) DC-PCR strategies. Only the outside primers used in nested approaches are shown. Paired transgene-specific primers Les1 and DC3 will amplify both sides of the palindrome. The side-specific DC-PCR uses one transgene and one chromosomal primer (Les1 and ACLF1 in the case of the left side and Les1 plus ALRF1 for the right side). A, ApaI; B, BamHI; E, EcoRI; P, PstI; (P), cleaved or former PstI site.

FIG. 3.

Germ line and somatic palindrome modifications are comparable. Sequences were determined by conventional PCR for panels C and D, by DC-PCR for panels A and E, and by both methods for panel B. For comparison, all deletions (shading) are shown on the left side; however, the deletion in panel B is actually on the right side, those of panels C and D are unknown, and that of E is on the left side. Underlining indicates short homologies that that can be assigned to either the left or the right side of a given deletion. Bold typeface indicates junctional insertions of undefined origin (possibly due to terminal deoxynucleotidyl transferase activity in the Ab-MLV cells; however, similar insertions have also been observed in nonlymphoid cells in other studies [21]). (A) Partial sequence of the intact central region of the Line 78 palindrome in test clones 7#7-7-6 and 7#7-7-11. The symmetry axis is indicated by the head-to-head arrows. (B) Inherited deletion arising in our colony (not detected on Southern blots). (C) Deletion in line 18#1. (D) Deletion in test clone 7#14-11. (E) Deletion in the sample subclone 7#7-7-6-22 (day 64). Deletions can also include the central PstI site (see Fig. 6 and examples in reference 22).

FIG. 4.

Analysis of rearrangements among 7#7-7-6 sample subclones (day 64). Cellular DNA was codigested with BamHI and PstI and was probed as indicated in the legend to Fig. 2. The subclone DNA in lanes 3, 5, 6, 7, 8, and 14 exhibited intact transgenes as indicated by 3.4- and 2.4-kb bands. The remaining subclones were rearranged, as was the subclone in lane 4, as discovered upon a subsequent EcoRI analysis (data not shown) (see Materials and Methods).

FIG. 5.

Lack of further rearrangement in 7#14-11. Some day 92 subclones from a culture of 7#14-11 are shown. No further rearrangements in any of the 105 subclones sampled from 7#14-11 were seen.

FIG. 6.

Rearrangement outcomes. (A) Three classes of outcome are defined according to structure: deletion subclones have restriction maps indicative of a central deletion, compound subclones have restriction maps indicative of a central deletion and also contain a homologously recombined lacZ copy (one subclone listed in this category had a central deletion undetected on Southern blots but revealed by DC-PCR), and recombinant subclones have restriction maps predicted for a straightforward homologous recombination and were confirmed in three of the five examples to lack central deletions by DC-PCR. The deduced mechanism and the number of subclones in each category as well as a reference to the appropriate diagrams in Fig. 7 and 8 are also given (202 other subclones were intact). Deletion subclones were subgrouped (a, b, c) according to the size of the central deletion as well as whether the central PstI site was or was not preserved. The compound and recombinant categories are each subgrouped according to whether the lacZ recombinant is 5′-inner-to-3′-outer (2.6-kb band, d and f) or 5′-outer-to-3′-inner (3.1-kb band, e and g.) Three subclones (other) had banding patterns that did not fit the criteria for any category (see the text for details). (B) Pie chart showing the proportions of each type of structure among the 118 rearranged subclones. (C) Examples of different types of patterns obtained with BamHI and PstI codigestion. Lanes are from four different gels. Adjustments were made in the composite so that reference intact lanes from each image (cropped out) would overlie.

FIG. 7.

Center-break activity can generate recombinant lacZ structures as well as simple deletions. Top, a center-break is created at the palindrome symmetry center. (A) The break is enlarged and then reconnected by NHEJ. Alignment of broken ends can sometimes be mediated by microhomologies at the joined termini followed by limited gap filling (indicated by the short displaced top strand and dashed arrow); however, events that occur completely independently of any microhomology are also seen, as in Fig. 3. (B) NHEJ with strand invasion (expansion). The terminus to one side of the center-break is degraded to within the inner lacZ repeat. The 3′ end can then invade an intact outer lacZ repeat on the other side of the break. Following extension, the newly elongated end peels off and is joined to its partner by NHEJ (with gap filling). (C) NHEJ with strand invasion (deletion). The terminus to one side of the center-break is degraded to within the outer lacZ repeat; thereafter, strand invasion, extension, and joining take place as described for panel B. The predicted restriction map is given for each outcome.

FIG. 8.

Structures generated by homologous exchange. (A) Three recombinant products from crossing over between inner and outer lacZ repeats (dashed lines) are theoretically detectable. (B) Recombination between lacZ repeats located on the same arm of the palindrome is deletional and is indicated by the appearance of a 3.1-kb band in addition to the 2.4- and 3.4-kb bands. (C) Recombination between inner and outer lacZ repeats on opposite arms of the palindrome results in inversion and yields a four-band pattern: 3.1- and 2.6-kb bands (recombinant) plus 2.4- and 3.4-kb bands (nonrecombinant). (D) Reciprocal exchange between sister chromatids (as diagrammed by the gray copy in panel A) would give one expanded chromosome with an increased number of lacZ repeats (and a 3.4-, 3.1-, 2.6-, 2.4-kb banding pattern) and one deleted product (with a 3.4-, 3.1-, 2.4-kb banding pattern) as diagrammed in panel B.