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Monitoring the Replication and Repair of Psoralen Induced Interstrand Crosslinks on Human Genomic DNA


Mutreja, Karun. Monitoring the Replication and Repair of Psoralen Induced Interstrand Crosslinks on Human Genomic DNA. 2017, University of Zurich, Faculty of Science.

Abstract

Interstrand crosslinks (ICL) represent highly cytotoxic lesions that covalently link two strand of DNA in a duplex, thus posing a threat to replication and transcription, two fundamental processes for life. ICL inducing agents are among the most widely used chemotherapeutics, but unfortunately cancer cells are able to repair ICL using Fanconi Anemia (FA) and Homologous Recombination (HR) proteins, thereby evading or limiting cell death. Based on biochemical work with plasmid DNA and cell-free extracts, ICL were long considered an absolute roadblock to the replication machinery, but a recent study in human cells has shown that the replication machinery can in fact efficiently traverse ICLs. We synthesized a chemically modified psoralen (DIG-TMP) that can be recognized by specific antibodies and exploited single-molecule approaches – i.e. mainly DNA fiber assays and visualization of replication intermediates by Electron Microscopy (EM) - to monitor directly human genome replication across ICLs (local replication), as well as elsewhere in the genome (global replication). Performing this parallel analysis in different genetic backgrounds (i.e. upon inactivation of the crucial FA factors FANCM, FANCD2 and RAD51, as well as the fork remodeling factor ZRANB3), we found that (i) the fork-remodeling factor RAD51 is immediately recruited to stressed replication forks before any detectable double stranded break formation, (ii) replication fork reversal is very frequent upon DIG-TMP treatment, assists ICL traverse and prevents premature SLX4-mediated chromosomal breakage, (iii) global fork slowdown upon ICL induction is associated with fork reversal and depends upon ATR activity. I also developed Immuno Electron Microscopy (iEM) to directly visualize ICLs on individual replication intermediates: albeit technically challenging and currently not applicable for large scale analysis, this approach showed potential to reveal architectural changes of replication forks facing ICLs and to assess a direct functional link between fork remodeling and ICL bypass or repair. This study provides important mechanistic insight on ICL replication and repair and contributes to clarify resistance mechanisms upon cancer chemotherapeutic regimens with ICL-inducing agents.

Abstract

Interstrand crosslinks (ICL) represent highly cytotoxic lesions that covalently link two strand of DNA in a duplex, thus posing a threat to replication and transcription, two fundamental processes for life. ICL inducing agents are among the most widely used chemotherapeutics, but unfortunately cancer cells are able to repair ICL using Fanconi Anemia (FA) and Homologous Recombination (HR) proteins, thereby evading or limiting cell death. Based on biochemical work with plasmid DNA and cell-free extracts, ICL were long considered an absolute roadblock to the replication machinery, but a recent study in human cells has shown that the replication machinery can in fact efficiently traverse ICLs. We synthesized a chemically modified psoralen (DIG-TMP) that can be recognized by specific antibodies and exploited single-molecule approaches – i.e. mainly DNA fiber assays and visualization of replication intermediates by Electron Microscopy (EM) - to monitor directly human genome replication across ICLs (local replication), as well as elsewhere in the genome (global replication). Performing this parallel analysis in different genetic backgrounds (i.e. upon inactivation of the crucial FA factors FANCM, FANCD2 and RAD51, as well as the fork remodeling factor ZRANB3), we found that (i) the fork-remodeling factor RAD51 is immediately recruited to stressed replication forks before any detectable double stranded break formation, (ii) replication fork reversal is very frequent upon DIG-TMP treatment, assists ICL traverse and prevents premature SLX4-mediated chromosomal breakage, (iii) global fork slowdown upon ICL induction is associated with fork reversal and depends upon ATR activity. I also developed Immuno Electron Microscopy (iEM) to directly visualize ICLs on individual replication intermediates: albeit technically challenging and currently not applicable for large scale analysis, this approach showed potential to reveal architectural changes of replication forks facing ICLs and to assess a direct functional link between fork remodeling and ICL bypass or repair. This study provides important mechanistic insight on ICL replication and repair and contributes to clarify resistance mechanisms upon cancer chemotherapeutic regimens with ICL-inducing agents.

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Additional indexing

Item Type:Dissertation
Referees:Lopes Massimo, Gari Kerstin, Constantinou Angelos, Esashi Fumikon
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:2017
Deposited On:06 Feb 2018 14:17
Last Modified:19 Mar 2018 10:26
OA Status:Closed

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