A two-step mechanism for palindrome-mediated rearrangements in human cells
Purpose: The t(11;22)(q23;q11) is a recurrent constitutional translocation in humans. Breakpoints on both chromosomes have been located within palindromic AT-rich repeats (PATRRs). We propose that these PATRRs form cruciform structures in vivo and induce DSBs that are illegitimately repaired through non-homologous end-joining (NHEJ). To elucidate how the PATRRs induce DSBs, we established a plasmid-based model using a mammalian cell line as host.
Method: We independently cloned the PATRRs from 11q23 and 22q11 into plasmids and co-transfected them into HEK293 cells. DNA was recovered and used for PCR or Southern analysis. A time-course for cleavage and rearrangement of the PATRRs was determined.
Result: By PCR we could detect rearranged molecules cleaved and rejoined at the PATRRs. Sequence analysis revealed that the majority of rearranged molecules were recombined at or near the center of the palindrome. Their structure was quite similar to that of human t(11;22) junction fragments. Rearranged PCR products appeared after ~5h, increased to a maximum at 36 h, and then gradually decreased. Southern analysis reveals that the plasmids appear to be cleaved at the center of the palindrome, i.e. at the tip of the cruciform. However, two distinct bands were observed when electrophoresed under alkaline condition, indicative of diagonal cleavage at the base of the putative cruciform structure. Further, presence of an additional larger band suggests sealing of the nick generated by diagonal breakage of the cruciform. All of these bands were detected within 1 h.
Conclusion: We propose that a two-step mechanism is responsible for PATRR-mediated translocations: 1) a rapid diagonal cleavage at the cruciform followed by nick-sealing, and 2) a slower hairpin opening by tip cleavage that is followed by repair through the NHEJ pathway between two open hairpins.