Cruciform DNA Structure Underlies the Etiology for Palindrome-mediated Human Chromosomal Translocations

Abstract

There is accumulating evidence to suggest that palindromic AT-rich repeats (PATRRs) represent hot spots of double-strand breakage that lead to recurrent chromosomal translocations in humans. As a mechanism for such rearrangements, we proposed that the PATRR forms a cruciform structure that is the source of genomic instability. To test this hypothesis, we have investigated the tertiary structure of a cloned PATRR. We have observed that a plasmid containing this PATRR undergoesaconformationalchange,causingtemperaturedependent mobility changes upon agarose gel electrophoresis. The mobility shift is observed in physiologic salt concentrations and is most prominent when the plasmid DNA is incubated at room temperature prior to electrophoresis. Analysis using two-dimensional gel electrophoresis indicates that the mobility shift results from the formation of a cruciform structure. S1 nuclease and T7 endonuclease both cut the plasmid into a linear form, also suggesting cruciform formation. Furthermore, anti-cruciform DNA antibody reduces the electrophoretic mobility of the PATRR-containing fragment. Finally, we have directly visualized cruciform extrusions from the plasmid DNA with the size expected of hairpin arms using atomic force microscopy. Our data imply that for human chromosomes, translocation susceptibility is mediated by PATRRs and likely results from their unstable conformation.The constitutional t(11;22)(q23;q11) is the only known recurrent non-Robertsonian translocation in humans. Its recurrent nature implicates a specific genomic structure at the t(11;22) breakpoints. Analyses of numerous independent t(11;22) cases have localized the breakpoints within palindromic AT-rich repeats (PATRRs) 1 on 11q23 and 22q11 (1-4). Most 11;22 translocations show breakpoints at the center of the PATRRs, suggesting that the center of the palindrome is susceptible to double-strand breaks, leading to the translocation (5, 6). Indeed, translocation-specific PCR detects a high frequency of de novo t(11;22)s in normal sperm samples (7).The breakpoints on 22q11 are located within one of the unclonable gaps in the human genome (8, 9). Extensive screening of YAC/BAC/PAC libraries has not been successful in cloning this breakpoint region. However, experimentally derived sequences from numerous t(11;22) junction fragments demonstrate that the 22q11 breakpoints reside within a larger PATRR. The breakpoints of a variety of translocations involving 22q11 cluster within this region, suggesting that the 22q11 PATRR is highly unstable (10 -13). More recently, molecular cloning of translocation breakpoints has demonstrated similar palindromic sequences on partner chromosomes, such as 17q11, 4q35

Self-catalyzed site-specific depurination of guanine residues within gene sequences

“…However, superhelical stress in vivo induces formation of stem-loops in duplex DNA at inverted repeats (19,20), and stems as short as 5-7 bp easily extrude under physiological superhelical stress (21).…”

Palindromic AT-rich repeat in theNF1gene is hypervariable in humans and evolutionarily conserved in primates

“…Palindromic regions form a cruciform in negatively supercoiled DNA by unwinding the negative superhelicity [Sinden, 1994]. When PATRR plasmids extrude cruciforms, they migrate as a ladder in standard agarose electrophoresis [Kurahashi et al, 2004]. When 17PATRR plasmids were examined, unwound plasmids migrating as a ladder were observed in all symmetric (17-L-PATRR (p-(AT) 10 ; d-(AT) 10 )) and asymmetric (17-L-PATRR (p-(AT) 10 ; d-(AT) 20 ); 17-S-PATRR) plasmids at lane N (Fig.…”

Functional Polymorphisms of the Lss and Fdft1 Genes in Laboratory Rats

“…4). DNA palindromes are a hot spot for deletions [5,9] or chromosomal translocations [7], and hence are frequently found as causes of human hereditary diseases. The molecules and precise mechanisms involved in the mutagenesis depicted in Fig.…”