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

Abstract

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 a ~300 basepair subregion (Flex1) of human CFS FRA16D in yeast, and found it recapitulated characteristics of CFS fragility in human cells. Flex1 fragility was 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, likely acting within an Slx1-4- Rad1-10 complex, while 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 of forks stalled at DNA structures. Furthermore, our results suggest that CFSs are not just prone to breakage but also impaired in their ability to heal, and this deleterious combination accounts for their fragility.

Keywords: common fragile site (CFS), FRA16D, cruciform structure, AT repeat, Mus81 endonuclease, Sae2/CtIP