Single Molecular Analysis of the Encounter of Replication Forks with DNA Interstrand Crosslinks

Wednesday, October 10, 2012 — Poster Session II
Noon – 2:00 p.m	Natcher Conference Center, Building 45	NIA	CELLBIO-2

* FARE Award Winner

Authors

• J Huang
• Y Wang
• S Liu
• M.M Seidman

Abstract

DNA interstrand crosslinks (ICLs) are absolute blocks to replication. Progress of ICL repair study, particularly at replication forks, has been limited by the lack of approaches for the direct analysis of replication in the vicinity of ICLs. Additionally, most crosslinking agents generate primarily monoadducts, with only a small fraction of actual ICLs, complicating interpretation of experiments with these compounds. We have developed a novel approach for single-molecule analysis of replication to study the encounter of individual replication forks with ICLs. We employed antigen tagged psoralen (dig-pso) which forms a high frequency of ICLs following photoactivation by ultraviolet light (UVA). Cells are treated with the dig-pso /UVA and then pulsed with halogenated thymidine derivatives. The location of replication tracts in the vicinity of ICLs can be determined by immunofluorescent visualization of the thymidine analogues and dig-pso on DNA fibers. Single fork collisions with the ICLs do occur, but replication on either side of the ICLs is observed more frequently. The double sided patterns are due to the collision of existing replicons, rather than activation of nearby dormant origins. Our data also suggest that removal of the ICLs is slow relative to the resumption of replication, arguing against a replication-repair-replication.

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