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Section on Gene Targeting

Michael Seidman, Ph.D., Chief

Actively dividing cells are continually challenged by impediments to the progression of replication forks. These include alternate DNA structures, breaks in template strands, and a broad range of chemical adducts resulting from exposure to sunlight and other environmental agents, as well as radical species generated internally by oxidative metabolism. Individuals with defects in the genes that encode factors that protect cells from replication stress typically suffer from severe clinical pathologies including cancer, neurodegeneration, features of accelerated aging, and early death. These genetic disorders emphasize the importance of a faithful and effective cellular response to replication challenge for   normal development, healthy life, and healthful aging.

Research in the Section on Gene Targeting is directed towards an understanding of how cells respond to replication stress imposed by DNA reactive compounds, with a focus on a particular structure- the interstrand crosslink (ICL). They can be formed by products of lipid peroxidation, abasic sugars, and by several important cancer chemotherapy drugs. ICLs engage both strands of DNA, present a profound challenge to replication and transcription, and thus are very toxic.

Portfolio/Research Areas

  • Cellular responses to replication stress
  • Replication independent repair of interstrand crosslinks
  • Role of the DNA Damage Response in cellular senescence

Findings and Publications

We have developed two novel strategies to support research on the cellular response to ICLs. In both programs we have exploited the properties of psoralen, a photoactive crosslinking agent. This compound is found in plants, intercalates in DNA and, upon exposure to long wave UV light (UVA), reacts with thymines on opposite strands at T:A and A:T sites, crosslinking the duplex. We synthesized an antigen tagged derivative which enables visualization of psoralen adducts in DNA by immunofluorescence. In one experimental series we have made use of laser confocal microscopy to introduce psoralen ICLs into defined subnuclear regions of individual live cells. We have monitored the persistence of the tagged psoralen as a function of the host cell genetics. We have also followed the recruitment of DNA Damage Response proteins to the localized ICLs, with a particular focus on the participation of the Fanconi Anemia proteins. These experiments are largely concerned with the activation of the DDR by localized ICLs independent of replication. Repair of ICLs lowers the lesion burden prior to replication, thus reducing the probability of fork encounters with these adducts.

However, replication fork collisions with ICLs do occur. In order to study these events we have designed a new approach based on DNA fiber assays combined with single molecule imaging with immunoquantum dots. In a typical experiment cells are treated with the antigen tagged psoralen/UVA and then pulsed with halogenated nucleoside analogues. The cells are harvested, DNA fibers are stretched on microscope slides, and the location of psoralen adducts displayed with an immunoquantum dot and the replication tracks illuminated by immunofluorescence. The patterns of the nucleoside analogue tracts in the vicinity of the quantum dots reflect the replication fork encounters with the ICLs. These and related experiments distinguish single fork encounters, in which a replication tracts stops at a psoralen site, from double fork encounters, in which two forks from opposite directions collide at an ICL. In addition, we discovered an unexpected pattern in which, following an encounter on one side, the replication fork restarts on the distal side of an intact ICL, continuing on in the same direction. These “traverse “patterns require the translocase activity of the Fanconi Anemia protein M. While replication restart on the distal side of an ICL takes a few minutes, the repair events that separate one strand from the other occur over several hours. Thus completion of replication appears to take precedence over removal of the DNA lesions.

Muniandy PA, Thapa D, Thazhathveetil AK, Liu ST, Seidman MM. Repair of laser localized DNA interstrand cross-links in G1 phase mammalian cells. J Biol Chem. 284, 27908-17, 2009

Zhijiang Yan, Rong Guo, Manikandan Paramasivam, Weiping Shen, Chen Ling, David Fox III, Yucai Wang, Anneke B. Oostra, Julia Kuehl, Duck-Yeon Lee, Minoru Takata, Maureen E. Hoatlin, Detlev Schindler, Hans Joenje, Johan P. de Winter, Lei Li, Michael M. Seidman, and Weidong Wang* A ubiquitin-binding protein FAAP20 links RNF8-mediated ubiquitination to the Fanconi anemia DNA Repair network.  Mol Cell, 47:61-75, 2012

Huang J, Liu S,Bellani MA, Thazhathveetil AK, Ling C, de Winter JP, Wang Y, Wang W, Seidman MM. The DNA Translocase FANCM/MHF Promotes Replication Traverse of DNA Interstrand Crosslinks. Mol.Cell 52, 434-446, 2013

Huang J, Gali H, Paramasivam M, Muniandy P, Gichimu J, Bellani MA, Seidman MM. Single Molecule Analysis of Laser Localized Interstrand Crosslinks. Front Genet. 7, 84, 2016

Ling C, Huang J, Yan Z, Li Y, Ohzeki M, Ishiai M, Xu D, Takata M, Seidman M, Wang W. Bloom syndrome complex promotes FANCM recruitment to stalled replication forks and facilitates both repair and traverse of DNA interstrand crosslinks. Cell Discov.  2, 16047, 2016