Transcription influences the types of deletion and expansion products in an orientation-dependent manner from GAC*GTC repeats

Abstract

The genetic instability of (GAC*GTC)n (where n = 6-74) was investigated in an Escherichia coli-based plasmid system. Prior work implicated the instability of a (GAC*GTC)5 tract in the cartilage oligomeric matrix protein (COMP) gene to the 4, 6 or 7mers in the etiology of pseudoachondroplasia and multiple epiphyseal dysplasia. The effects of triplet repeat length and orientation were studied after multiple replication cycles in vivo. A transcribed plasmid containing (GAC*GTC)49 repeats led to large deletions (>3 repeats) after propagation in E.coli; however, if transcription was silenced by the LacI(Q) repressor, small expansions and deletions (<3 repeats) predominated the mutation spectra. In contrast, propagation of similar length but opposing orientation (GTC*GAC)53 containing plasmid led to small instabilities that were unaffected by the repression of transcription. Thus, by inhibiting transcription, the genetic instability of (GAC*GTC)49 repeats did not significantly differ from the opposing orientation, (GTC*GAC)53. We postulate that small instabilities of GAC*GTC repeats are achieved through replicative slippage, whereas large deletion events are found when GAC*GTC repeats are transcribed. Herein, we report the first genetic study on GAC*GTC repeat instability describing two types of mutational patterns that can be partitioned by transcription modulation. Along with prior biophysical data, these results lay the initial groundwork for understanding the genetic processes responsible for triplet repeat mutations in the COMP gene.

Figure 1

Analyses of in vivo genetic instabilities of (GAC•GTC)_n and (GTC•GAC)_n repeats. The results of a representative recultivation experiment through four growth cycles (indicated as 1–4) in E.coli AB1157 are shown; the plasmid DNA products were digested with HindIII and SacI, and the fragments, radiolabeled with [³²-P]dATP, were separated on a native 7% acrylamide gel as described under Materials and Methods. The arrowheads indicate the full-length fragments (no expansions or deletions) for each parental plasmid. The products of expansions and deletions are shown as a function of the growth cycles of plasmids containing (A) (GAC•GTC)₂₇ and (GTC•GAC)₃₀ repeats (B) (GAC•GTC)₄₉ and (GTC•GAC)₅₃ repeats and (C) (GAC•GTC)₇₄ and (GTC•GAC)₆₉ repeats.

Figure 2

The amounts of products formed from (GAC•GTC)_n and (GTC•GAC)_n repeats as a function of growth cycles. The extents of genetic instabilities were determined by quantitating the areas of all peaks in each lane as described in Materials and Methods. The averages from three independent recultivation experiments (including experiments from Figure 1) of percentage expansions (filled bars), deletions (gray bars) and original length fragments (crosshatched bars) are plotted against the number of growth cycles (indicated as 1–4). The length and orientation of the inserts are denoted above each plot.

Figure 3

The effects of transcription silencing on the genetic instabilities of (GAC•GTC)₄₉ and (GTC•GAC)₅₃ repeats. E.coli AB1157 harboring pI^Q-kan was transformed with the supercoiled monomers of the designated plasmids. The instability of the (GAC•GTC)₄₉ and (GTC•GAC)₅₃ inserts were analyzed as described in the legend to Figure 1 and in Materials and Methods. The growth cycles are indicated as 1–4. The full-length progenitor fragments of the each parental plasmid are indicated by an arrowhead. (A) The results from a representative recultivation experiment of pRW4608 and pRW3820 that were carried out in the presence of LacI^Q repressor for determining the extent of genetic instabilities derived from replication-mediated events while lacZ gene transcription was inhibited. (B) The results from a recultivation experiment with pRW4608 and pRW3820 as a function of four growth cycles in the presence LacI^Q repressor and IPTG.

Figure 4

The effects of transcription silencing on the percentage of genetic instabilities of (GAC•GTC)₄₉ and (GTC•GAC)₅₃ repeats. Instability studies similar to the data in Figure 3 were quantitated as described in Materials and Methods. (A) The extent of instabilities of pRW4608 and pRW3820 in E.coli AB1157 in the presence of the LacI^Q repressor is expressed by percentages of expansions (filled bars), deletions (gray bars) and original full-length material (crosshatched bars) and plotted against multiple growth cycles (indicated as 1–4). (B) The percentages of genetic instabilities of pRW4608 and pRW3820 when transcription is restored by the addition of IPTG and LacI^Q repressor. The percentages are averages of three individual experiments including Figure 3.

Figure 5

The effects of transcription silencing on the longer repeat tracts of (GAC•GTC)₇₄ and (GTC•GAC)₆₉. E.coli AB1157 harboring pI^Q-kan was transformed with pRW3815 and pRW4607 for determining the influence of transcription through longer TRS repeats. (A) Through four recultivations, the DNAs were isolated and the TRS-containing inserts were released with HindIII and SacI digestion. The figure shows a 7% native gel electropherogram of the products through four growth cycles (indicated as 1–4) when transcription was inhibited by the LacI^Q repressor. Arrowheads mark the full-length fragments derived from the parental plasmids. (B) The percentage of genetic instability is displayed for pRW3815 and pRW4607 through the four growth cycles. The expansions (filled bars), deletions (gray bars) and original progenitor full-length fragments (crosshatched bars) are plotted against the number of growth cycles. The percentages are averages of three individual experiments including the data in (A).

Figure 6

Model for DNA slippage during replication of GAC•GTC and GTC•GAC repeats when transcription is silenced. During DNA replication, the lagging strand synthesis allows for single-stranded regions to misalign, either on the lagging strand template (A) or on the lagging nascent strand (B) in the regions of the repeating tract. (A) While in (a), the leading strand template (top strand of fork) retains the progenitor length (N), in (b) DNA slippage occurs by one repeat on the lagging strand. The DNA polymerase complex is proposed to bypass the looped-out repeat on the lagging strand template (17). (c) In the subsequent replication cycle, the new leading strand template has a deletion of 1 repeat (N − 1) whereas the new lagging strand template retains the progenitor repeat length (N). (B) (a) is the same as described for (A). (d) If the slippage of 1 repeat occurs on the nascent lagging strand, (e) then the newly synthesized top strand in the subsequent replication cycle will have an expansion of one repeat (N + 1) while the lower lagging strand template retains the progenitor length (N) (17,24).

Figure 7

Model for the involvement of transcription in the instabilities of GAC•GTC and GTC•GAC repeats of an intermediate length. RNA polymerase causes local duplex melting at the TRS tract for the initiation of transcription (40). Local positive and negative superhelical domains aid the progression of the RNA polymerase through the duplex DNA (41,50). While the transcribed GAC strand (left panel) complexes with the newly formed mRNA, the GTC strand which is not transcribed can readily form a hairpin. Re-annealing of the duplex DNA after transcription termination allows for the remaining unpaired bases on the GAC strand to loop out. The hairpin or looped-out structures are potential substrates for DNA repair proteins such as those that are involved in MMR (43), NER (51,52) or the SOS response (43). It is proposed that the structures are excised, DNA polymerase fills-in the gaps and the nicks are sealed by ligase. The removal of several bases due to their engagement as a hairpin or looped-out structure leads to large deletions on both strands of the DNA. In the other TRS orientation (right panel), when the GTC strand pairs with the mRNA, the GAC strand becomes unpaired; however, the GAC strand at an intermediate length does not form a hairpin structure as readily as the GTC unpaired strand (left panel). Therefore, transcription through intermediate-length GTC•GAC repeats does not stimulate genetic instabilities.