Rad18 is required for functional interactions between FANCD2, BRCA2, and Rad51 to repair DNA topoisomerase 1-poisons induced lesions and promote fork recovery.

Kaushlendra Tripathi,David W Clark,Chinnadurai Mani,Komaraiah Palle

doi:10.18632/oncotarget.7247

Abstract

Camptothecin (CPT) and its analogues are chemotherapeutic agents that covalently and reversibly link DNA Topoisomerase I to its nicked DNA intermediate eliciting the formation of DNA double strand breaks (DSB) during replication. The repair of these DSB involves multiple DNA damage response and repair proteins. Here we demonstrate that CPT-induced DNA damage promotes functional interactions between BRCA2, FANCD2, Rad18, and Rad51 to repair the replication-associated DSB through homologous recombination (HR). Loss of any of these proteins leads to equal disruption of HR repair, causes chromosomal aberrations and sensitizes cells to CPT. Rad18 appears to function upstream in this repair pathway as its downregulation prevents activation of FANCD2, diminishes BRCA2 and Rad51 protein levels, formation of nuclear foci of all three proteins and recovery of stalled or collapsed replication forks in response to CPT. Taken together this work further elucidates the complex interplay of DNA repair proteins in the repair of replication-associated DSB.

Highlights

DNA topoisomerase 1 (Top1) is an essential enzyme in higher eukaryotes, which resolves topological barriers during most critical cellular processes involving DNA, including replication, transcription, recombination and repair [1,2,3,4]
Rad18 is required for proper FANCD2, BRCA2, and Rad51 foci formation in response to CPTinduced double strand breaks (DSB)
Rad18 is important for efficient activation of the Fanconi anemia (FA) pathway; the functional interplay between Rad18 status and FANCD2, BRCA2 and Rad51 proteins in repair of replicationassociated DNA lesions is not known

Summary

Introduction

DNA topoisomerase 1 (Top1) is an essential enzyme in higher eukaryotes, which resolves topological barriers during most critical cellular processes involving DNA, including replication, transcription, recombination and repair [1,2,3,4]. The covalently bound Top1cc is transient and mostly undetectable because religation is much faster than cleavage in Top catalysis [3, 4] Anticancer agents such as camptothecin (CPT) and its clinical analogues Collision between an active replication fork and the Top1cc is capable of generating DNA double strand breaks (DSB) which can introduce mutations or lead to cell death [13,14,15] Anticancer agents, such as DNA Top poisons as well as others that result in stalled replication forks (hydroxyurea, gemcitabine, and others) are effective as they lead to high levels of DSB in rapidly replicating cancer cells as opposed to quiescent terminally differentiated normal cells [14, 16, 17]. Rad51) as well as associated proteins such as Rad, members of the Fanconi anemia (FA) family and the breast cancer associated (BRCA1, BRCA2) proteins sensitize cells to CPT [24,25,26,27,28]

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