Repair of localized DNA damage

National Institute On Aging

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Abstract

Failure to respond effectively to replication stress is recognized as a key contributor to developmental defects, premature aging syndromes, and the development of neoplasias. We have exploited the properties of interstrand crosslinks (ICLs) to develop a novel approach to examine the consequences of replisome encounters with a potent block. We synthesized an antigen tagged version of psoralen, a photoactive DNA crosslinking compound. We have combined well established procedures for displaying replication tracts on DNA fibers with immuno-quantum dot detection of individual antigen tagged psoralen ICLs. We observed single and double fork collisions as well as an unanticipated pattern of DNA synthesis on the side distal to the encounter. We termed this replication traverse of the ICLs and found that it is the major pattern. The DNA translocase, FANCM, is required for the traverse pathway. However, these events are independent of the Fanconi Anemia (FA) core complex proteins that ubiquitylate FANCD2, the central protein of the FA pathway. Since the FA proteins appear in vertebrate lineages, while FANCM is found in Archaea, we propose that the traverse pathways evolved early in response to major replication challenges. Notably, non-ubiquitinated FANCD2 is also required for traverse and is epistatic with FANCM. FANCM associates with a subset of replisomes that encounter the block. These replisomes lose one of the key components of the replication apparatus as a result of binding FANCM. Consequently, there is a FANCM dependent remodeling of the replisome. Another subset of replisomes that encounter the block are associated with the recently described DONSON protein. The DONSON replisomes are active in euchromatin and show a corresponding bias towards early replicating regions. The FANCM associated replisomes are skewed towards heterochromatin and late replicating sequences. The two replisome species are distinct from one another. FANCM does not appear on DONSON replisomes and DONSON does not appear on FANCM replisomes. The association of the DNA translocase FANCM with the heterochromatin replisome suggested that there was a corresponding translocase associated with the euchromatin replisome. We have now identified this translocase. Consequently while there is a compositional distinction between the two replisomes there is a functional symmetry between them. Our results reveal an unanticipated complexity in the cellular response to replication stress. They raise important questions about the response to replication stress in cells in aged individuals in which there is a decline in heterochromatin and transcriptional activation of previously silenced sequences. We are now addressing these questions in proliferating B cells isolated from young and old donors.