Search for: All records

Abstract Alternative non‐B form DNA structures, also called secondary structures, can form in certain DNA sequences under conditions that produce single‐stranded DNA, such as during replication, transcription, and repair. Direct links between secondary structure formation, replication fork stalling, and genomic instability have been found for many repeated DNA sequences that cause disease when they expand. Common fragile sites (CFSs) are known to be AT‐rich and break under replication stress, yet the molecular basis for their fragility is still being investigated. Over the past several years, new evidence has linked both the formation of secondary structures and transcription to fork stalling and fragility of CFSs. How these two events may synergize to cause fragility and the role of nuclease cleavage at secondary structures in rare and CFSs are discussed here. We also highlight evidence for a new hypothesis that secondary structures at CFSs not only initiate fragility but also inhibit healing, resulting in their characteristic appearance. 

Expanded CAG/CTG repeats are sites of DNA damage, leading to changes in repeat length. To determine how ssDNA gap filling affects repeat instability, we inserted (CAG)70 or (CTG)70 repeats into a single-strand annealing (SSA) assay system such that resection and filling in the ssDNA gap would occur across the repeat tract. After resection, when the CTG sequence was the single-stranded template for fill-in synthesis, repeat contractions were elevated and the ssDNA created a fragile site that led to large deletions involving flanking homologous sequences. In contrast, resection was inhibited when CTG was on the resected strand, resulting in repeat expansions. Deleting Rad9, the ortholog of 53BP1, rescued repeat instability and lost viability by increasing resection and fill-in speed. Deletion of Rad51 increased CTG contractions and decreased survival, implicating Rad51 in protecting ssDNA during gap filling. Taken together, DNA sequence within a single-stranded gap determines repair kinetics, fragility, and repeat instability. 

Trinucleotide repeats are common in the human genome and can undergo changes in repeat number and cause length-dependent chromosome fragility. Expanded CAG repeats have been linked to over 14 human diseases and are considered hotspots for breakage and genomic rearrangement. Here we describe two Saccharomyces cerevisiae based assays that evaluate the rate of chromosome breakage that occurs within a repeat tract (fragility), with variations that allow the role of transcription to be evaluated. The first fragility assay utilizes end-loss and subsequent telomere addition as the main mode of repair of a yeast artificial chromosome (YAC). The second fragility assay relies on the fact that a chromosomal break stimulates recombination-mediated repair. A PCR-based assay can be used to evaluate instability of the repeat in the same conditions used to measure repeat fragility. These assays have contributed to understanding the genetic mechanisms that cause chromosome breaks and tract-length changes at unstable trinucleotide repeats.