Repair of localized DNA damage

不能有效地应对复制应激被认为是发育缺陷、早衰综合征和肿瘤发生的关键因素。我们利用链间交联（ICLs）的性质，开发了一种新的方法来研究复制体遇到一个有效的块的后果。我们合成了一种抗原标记的peptide，一种光敏DNA交联化合物。我们已经结合了完善的程序显示复制道的DNA纤维与免疫量子点检测个别抗原标记的peptide ICLs。我们观察到单叉和双叉碰撞，以及在遭遇远端一侧的意外DNA合成模式。我们称之为ICL的复制遍历，并发现这是主要的模式。DNA移位酶FANCM是横向途径所必需的。然而，这些事件是独立的范可尼贫血（FA）的核心复合物蛋白泛素化FANCD 2，FA途径的中心蛋白。由于FA蛋白出现在脊椎动物谱系中，而FANCM发现于脊椎动物中，我们提出，遍历途径在早期进化以应对主要的复制挑战。值得注意的是，非泛素化的FANCD 2也是遍历所需的，并且与FANCM上位。 FANCM与遇到阻断的复制体的子集相关联。由于结合FANCM，这些复制体失去了复制装置的关键组件之一。因此，存在FANCM依赖的复制体重塑。遇到阻断的另一个复制体子集与最近描述的DONSON蛋白相关。DONSON复制体在常染色质中是活跃的，并显示出对早期复制区域的相应偏好。FANCM相关的复制体偏向异染色质和晚期复制序列。这两种复制体物种彼此不同。FANCM不出现在DONSON复制体上，DONSON不出现在FANCM复制体上。 DNA移位酶FANCM与异染色质复制体的关联表明存在与常染色质复制体相关联的相应的移位酶。我们现在已经鉴定了这种移位酶。因此，虽然两个复制体之间存在组成上的区别，但它们之间存在功能上的对称性。我们还鉴定了在异染色质复制体上未发现的与DONSON复制体相关的其他蛋白质。异染色质复制体也是感兴趣的。我们已经鉴定了几种对该复制体特异的蛋白质。 我们的研究结果揭示了一个意想不到的复杂性，在细胞反应复制压力。他们提出了重要的问题，在老年人的细胞中，其中有异染色质和转录激活以前沉默的序列下降的复制应激反应。我们现在正在从年轻和老年供体分离的增殖B细胞中解决这些问题。

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.