Sequence and Nuclease Requirements for Breakage and Healing of a Structure-Forming (AT)n Sequence within Fragile Site FRA16D

• Published in: Cell reports (Cambridge) 2019-04, Vol.27 (4), p.1151-1164.e5
• Main Authors: Kaushal, Simran, Wollmuth, Charles E., Das, Kohal, Hile, Suzanne E., Regan, Samantha B., Barnes, Ryan P., Haouzi, Alice, Lee, Soo Mi, House, Nealia C.M., Guyumdzhyan, Michael, Eckert, Kristin A., Freudenreich, Catherine H.
• Format: Article
• Language: English
• Subjects: AT repeat; CFS; Chromosome Breakage; Chromosome Fragile Sites - genetics; Chromosomes, Human, Pair 16 - genetics; common fragile site; cruciform structure; CtIP; DNA Polymerase III - genetics; DNA Polymerase III - metabolism; DNA Replication; Endonucleases - metabolism; FRA16D; Humans; Mus81 endonuclease; replication fork stall; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Sae2; Slx4 complex; Tandem Repeat Sequences
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2019.03.103

Common fragile sites (CFSs) are genomic regions that display gaps and breaks in human metaphase chromosomes under replication stress and are often deleted in cancer cells. We studied an ∼300-bp subregion (Flex1) of human CFS FRA16D in yeast and found that it recapitulates characteristics of CFS fragility in human cells. Flex1 fragility is dependent on the ability of a variable-length AT repeat to form a cruciform structure that stalls replication. Fragility at Flex1 is initiated by structure-specific endonuclease Mus81-Mms4 acting together with the Slx1-4/Rad1-10 complex, whereas Yen1 protects Flex1 against breakage. Sae2 is required for healing of Flex1 after breakage. Our study shows that breakage within a CFS can be initiated by nuclease cleavage at forks stalled at DNA structures. Furthermore, our results suggest that CFSs are not just prone to breakage but also are impaired in their ability to heal, and this deleterious combination accounts for their fragility. [Display omitted] •FRA16D subregion Flex1 causes fragility in an AT length-dependent manner•Longer AT repeats form a cruciform and stall replication by human Pol δ•Flex1 is targeted by the Mus81 nuclease, whereas Yen1 protects against breakage•Fragility at Flex1 is due to a combination of increased breaks and impaired healing Kaushal et al. found that a subregion of fragile site FRA16D forms an abnormal DNA structure that stalls DNA polymerase, resulting in DNA breakage and difficulty healing. They identify nucleases that cleave this structure to cause the DNA breaks and postulate that difficulty healing makes these sites prone to rearrangements.