Section on Gene Targeting

Michael Seidman, Ph.D., Chief

Actively dividing cells are continually challenged by impediments (referred to as replication stress) to the progression of replication forks. These include alternate DNA structures, breaks in template strands, and an extensive collection of chemical adducts resulting from exposure to sunlight, other environmental agents, and radical species generated internally by oxidative metabolism. Furthermore, the replication apparatus must replicate the entire genome through transcriptionally active euchromatin and heterochromatin which contains many “difficult to replicate” sequences. 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.

The essentials of the replication machinery are a helicase to unwind DNA and DNA polymerases to synthesize daughter DNA. The helicase component forms a locked ring around the template for leading strand synthesis, and once activated in S phase cannot disassemble and relocate. Stalled replication forks are sources of DNA fragments that can transit to the cytoplasm and induce the interferon responses, contributing to the persistent inflammation that underlies so many of the adversities of aging. Although the passage from genome to cytoplasm has attracted considerable attention, there is a much more limited understanding of the determinants of replisome stalling vs continued progression following an encounter with a block. Furthermore, the relationship between replisomes, chromatin architecture, and replication stress has received little attention.

Portfolio/Research Areas

• Cellular responses to replication stress
• Replisome remodeling

Findings and Publications

To address the fundamental question: what happens to the replisome when it encounters a fork stalling block, we developed a novel approach by exploiting a DNA structure, the interstrand crosslink (ICL), traditionally considered an absolute block to the replication apparatus. To visualize replication fork encounters with ICLs we took advantage of psoralen, a photoactive crosslinking agent that intercalates into DNA. Upon exposure to long wave UV light (UVA), it reacts with thymines on opposite strands at T:A and A:T sites, crosslinking the duplex.  Psoralens are amenable to modification without compromise to the crosslinking activity. We synthesized an antigen tagged conjugate which enables visualization of psoralen adducts in DNA by immunofluorescence. We utilized DNA fiber assays combined with single molecule imaging of the tagged ICLs to display the outcomes of replication fork collisions with the block. 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, 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 psoralen signals reflect the replication fork encounters with the ICLs. These experiments displayed single fork encounters, in which a replication tract stops at a psoralen site, and double fork encounters, in which two forks from opposite directions collide at an ICL. Unexpectedly, we discovered a pathway, that we termed replication traverse, that enabled restart of replication past the block. The new pathway was partially dependent on the activity of the FANCM DNA translocase. We found that FANCM associated with the replisome upon the encounter with the blocking lesion. Additionally, there was a FANCM dependent loss of proteins that lock the replisome onto the template for leading strand synthesis. Notably, the replication restart on the distal side of an ICL takes a few minutes, while 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.

Recently, we discovered another protein that is important for the traverse pathway. This protein, DONSON, also associates with replisomes that encounter ICLs, with the loss of the locking proteins. However, we found, again unexpectedly, that replisomes associated with DONSON were different and separable from those that bound FANCM. The two forms of the “stressed” replisome were also present in cells that had not been exposed to the blocking agent, likely the result of endogenous blocks in the genome. Furthermore, they could be distinguished by chromatin domain and replication timing. The FANCM replisome was preferentially found in heterochromatin and associated with telomeres and other late replicating sequences, while the other replisome was euchromatic and interacted with early replicating elements. These observations raise the question of how the erosion of chromatin distinctions during aging influences the cellular response to replication stress. This is of particular interest for the late replicating, normally heterochromatic, regions, the location of sequences that are the most difficult to replicate, and the most likely to present naturally occurring barriers to the replisome. The answer to these questions is the focus of the current work of the Section.

Selected References

1.   Thazhathveetil AK, Liu ST, Indig FE, Seidman MM. Psoralen conjugates for visualization of genomic interstrand cross-links localized by laser photoactivation. Bioconjug Chem. 2007;18(2):431-7.

2.   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. 2009;284(41):27908-17.

3.  Muniandy PA, Liu J, Majumdar A, Liu ST, Seidman MM. DNA interstrand crosslink repair in mammalian cells: step by step. Crit Rev Biochem Mol Biol. 2010;45(1):23-49.

4.  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. 2013;52(3):434-46. doi: S1097-2765(13)00713-2 [pii];10.1016/j.molcel.2013.09.021 [doi].

5.  Huang J, Gali H, Paramasivam M, Muniandy P, Gichimu J, Bellani MA, Seidman MM. Single Molecule Analysis of Laser Localized Interstrand Crosslinks. Front Genet. 2016;7:84. doi: 10.3389/fgene.2016.00084 [doi].

6.  Huang J, Zhang J, Bellani MA, Pokharel D, Gichimu J, James RC, Gali H, Ling C, Yan Z, Xu D, Chen J, Meetei AR, Li L, Wang W, Seidman MM. Remodeling of Interstrand Crosslink Proximal Replisomes Is Dependent on ATR, FANCM, and FANCD2. Cell Rep. 2019;27(6):1794-808. doi: S2211-1247(19)30497-8 [pii];10.1016/j.celrep.2019.04.032 [doi].

7.  Zhang J, Bellani MA, James RC, Pokharel D, Zhang Y, Reynolds JJ, McNee GS, Jackson AP, Stewart GS, Seidman MM. DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain. Nat Commun. 2020;11(1):3951. Epub 2020/08/10. doi: 10.1038/s41467-020-17449-1. PubMed PMID: 32769987.