Interconverting conformations of slipped-DNA junctions formed by trinucleotide repeats affect repair outcome.

Expansions of (CTG)·(CAG) repeated DNAs are the mutagenic cause of 14 neurological diseases, likely arising through the formation and processing of slipped-strand DNAs. These transient intermediates of repeat length mutations are formed by out-of-register mispairing of repeat units on complementary strands. The three-way slipped-DNA junction, at which the excess repeats slip out from the duplex, is a poorly understood feature common to these mutagenic intermediates. Here, we reveal that slipped junctions can assume a surprising number of interconverting conformations where the strand opposite the slip-out either is fully base paired or has one or two unpaired nucleotides. These unpaired nucleotides can also arise opposite either of the nonslipped junction arms. Junction conformation can affect binding by various structure-specific DNA repair proteins and can also alter correct nick-directed repair levels. Junctions that have the potential to contain unpaired nucleotides are repaired with a significantly higher efficiency than constrained fully paired junctions. Surprisingly, certain junction conformations are aberrantly repaired to expansion mutations: misdirection of repair to the non-nicked strand opposite the slip-out leads to integration of the excess slipped-out repeats rather than their excision. Thus, slipped-junction structure can determine whether repair attempts lead to correction or expansion mutations.

Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats.

R-loops have been described at immunoglobulin class switch sequences, prokaryotic and mitochondrial replication origins, and disease-associated (CAG)n and (GAA)n trinucleotide repeats. The determinants of trinucleotide R-loop formation are unclear. Trinucleotide repeat expansions cause diseases including DM1 (CTG)n, SCA1 (CAG)n, FRAXA (CGG)n, FRAXE (CCG)n and FRDA (GAA)n. Bidirectional convergent transcription across these disease repeats can occur. We find R-loops formed when CTG or CGG and their complementary strands CAG or CCG were transcribed; GAA transcription, but not TTC, yielded R-loops. R-loop formation was sensitive to DNA supercoiling, repeat length, insensitive to repeat interruptions, and formed by extension of RNA:DNA hybrids in the RNA polymerase. R-loops arose by transcription in one direction followed by transcription in the opposite direction, and during simultaneous convergent bidirectional transcription of the same repeat forming double R-loop structures. Since each transcribed disease repeat formed R-loops suggests they may have biological functions.

Repeat instability: mechanisms of dynamic mutations.

Disease-causing repeat instability is an important and unique form of mutation that is linked to more than 40 neurological, neurodegenerative and neuromuscular disorders. DNA repeat expansion mutations are dynamic and ongoing within tissues and across generations. The patterns of inherited and tissue-specific instability are determined by both gene-specific cis-elements and trans-acting DNA metabolic proteins. Repeat instability probably involves the formation of unusual DNA structures during DNA replication, repair and recombination. Experimental advances towards explaining the mechanisms of repeat instability have broadened our understanding of this mutational process. They have revealed surprising ways in which metabolic pathways can drive or protect from repeat instability.

Role of replication and CpG methylation in fragile X syndrome CGG deletions in primate cells.

Instability of the fragile X CGG repeat involves both maternally derived expansions and deletions in the gametes of full-mutation males. It has also been suggested that the absence of aberrant CpG methylation may enhance repeat deletions through an unknown process. The effect of CGG tract length, DNA replication direction, location of replication initiation, and CpG methylation upon CGG stability were investigated using an SV40 primate replication system. Replication-dependant deletions with 53 CGG repeats were observed when replication was initiated proximal to the repeat, with CGG as the lagging-strand template. When we initiated replication further from the repeat, while maintaining CGG as the lagging-strand template or using CCG as the lagging-strand template, significant instability was not observed. CpG methylation of the unstable template stabilized the repeat, decreasing both the frequency and the magnitude of deletion events. Furthermore, CpG methylation slowed the efficiency of replication for all templates. Interestingly, replication forks displayed no evidence of a block at the CGG repeat tract, regardless of replication direction or CpG methylation status. Templates with 20 CGG repeats were stable under all circumstances. These results reveal that CGG deletions occur during replication and are sensitive to replication-fork dynamics, tract length, and CpG methylation.

Effect of CAT or AGG interruptions and CpG methylation on nucleosome assembly upon trinucleotide repeats on spinocerebellar ataxia, type 1 and fragile X syndrome.

Nucleosome packaging regulates many aspects of DNA metabolism and is thought to mediate genetic instability and transcription of expanded trinucleotide repeats. Both instability and transcription are sensitive to repeat length, tract purity, and CpG methylation. CAT or AGG interruptions within the (CAG)n or (CGG)n tracts of spinocerebellar ataxia, type 1 or fragile X syndrome, respectively, confer increased genetic stability to the repeats. We report the formation of nucleosomes on sequences containing pure and interrupted (CAG)n and (CGG)n repeats having lengths above and below the genetic stability thresholds. Increased lengths of pure repeats led to increased and decreased propensities for nucleosome assembly on the (CAG)n and (CGG)n repeats, respectively. CpG methylation of the CGG repeat further reduced assembly. CAT interruptions in (CAG)n tracts decreased nucleosome assembly. In contrast, AGG interruptions in (CGG)n tracts did not affect assembly by hypoacetylated histones. The latter observation was unaltered by CpG methylation of the repeats. However, nucleosome assembly by hyperacetylated histones on interrupted CGG tracts was increased relative to pure tracts and this effect was abolished by CpG methylation. Thus, CAT or AGG interruptions can modulate the ability of (CAG)n and (CGG) tracts to assemble into chromatin and the effect of the AGG interruptions is dependent upon both the methylation status of the DNA and the acetylation status of the histones. Compared with the genetically unstable pure repeats, both interruptions permit a propensity of nucleosome assembly closer to that of random (genetically stable) sequences, suggesting an association of nucleosome assembly of trinucleotide repeats and genetic instability.