Repair of localized DNA damage

不能有效地应对复制压力被认为是发育缺陷、早衰综合征和肿瘤发展的关键因素。我们利用链间交联（ICLs）的特性，开发了一种新的方法来检查复制体与有效块相遇的后果。我们合成了一种抗原标记的补骨脂素，一种光活性DNA交联化合物。我们结合了在DNA纤维上显示复制束的完善程序和单个抗原标记的补骨脂素icl的免疫量子点检测。我们观察到单叉和双叉碰撞以及意想不到的DNA合成模式远端的遭遇。我们称之为icl的这种复制遍历，并发现这是主要模式。DNA转位酶，FANCM，是穿越途径所必需的。然而，这些事件与Fanconi贫血（FA）核心复合物蛋白无关，该蛋白泛素化FANCD2，即FA通路的中心蛋白。由于FA蛋白出现在脊椎动物谱系中，而FANCM蛋白则出现在古生菌中，因此我们提出，这些穿越途径在早期进化是为了应对主要的复制挑战。值得注意的是，非泛素化的FANCD2也是遍历所必需的，并且与FANCM具有上位性。

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. We have also identified additional proteins that are associated with the DONSON replisome that are not found on the heterochromatin replisome. The heterochromatin replisome is also of interest. We have identified several proteins that are specific to this replisome. 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.