Identification of Specific Inhibitors of Human RAD51 Recombinase Using High-Throughput Screening

Huang, Fei; Motlekar, Nuzhat; Burgwin, Chelsea M.; Napper, Andrew D.; Diamond, Scott L.; Mazin, Alexander V.

doi:10.1021/cb100428c

Cited by 197 publications

(198 citation statements)

References 30 publications

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“…Interestingly, upon differentiation, Rad51 binding to HPV DNA increased per viral genome, suggesting the formation of Rad51 nucleofilaments. To confirm the importance of Rad51 activity in productive viral replication, we utilized the Rad51-specific small-molecule inhibitor B02, which blocks Rad51 binding to DNA and inhibits DNA strand exchange into homologous sequences (47,48). As demonstrated in Fig.…”

Section: Inhibition Of Rad51 Function Prevents Productive Viral Replimentioning

confidence: 99%

Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

Chappell

Gautam

et al. 2016

J Virol

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High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. IMPORTANCEProductive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral replication, our findings suggest that HPV may utilize ATM activity to ensure localization of recombination factors to productively replicating viral genomes. The finding that E7 increases the levels of Rad51 and BRCA1 suggests that E7 contributes to productive replication by providing DNA repair factors required for viral DNA synthesis. Our studies not only imply a role for recombination in the regulation of productive HPV replication but provide further insight into how HPV manipulates the DDR to facilitate the productive phase of the viral life cycle. H uman papillomaviruses (HPVs) are small double-stranded DNA viruses approximately 8 kb in size that exhibit a preferential tropism for epithelial cells. High-risk mucosal HPV subtypes are the causative agents of cervical cancer and have been increasingly associated with anogenital, oropharyngeal, and head and neck cancers (1). The life cycle of HPV is intimately linked to the differentiation of its host cell, the keratinocyte (2). After exposure through a microwound in the stratified ep...

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Section: Inhibition Of Rad51 Function Prevents Productive Viral Replimentioning

confidence: 99%

Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

Chappell

Gautam

et al. 2016

J Virol

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“…Inactivation of these two enzymes or abolition of their interaction could then become a useful strategy to prevent DNA amplification and deletion through HR. In humans, some RAD51 inhibitors were identified by high-throughput screening of the NIH Small Molecule Repository (Ͼ200,000 compounds) (280). Specific RAD51 inhibitors for trypanosomatids could be found by this type of screening.…”

Section: Resistance and Treatmentmentioning

confidence: 99%

DNA Repair Pathways in Trypanosomatids: from DNA Repair to Drug Resistance

Genois

Paquet

Laffitte

et al. 2014

Microbiol Mol Biol Rev

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SUMMARY All living organisms are continuously faced with endogenous or exogenous stress conditions affecting genome stability. DNA repair pathways act as a defense mechanism, which is essential to maintain DNA integrity. There is much to learn about the regulation and functions of these mechanisms, not only in human cells but also equally in divergent organisms. In trypanosomatids, DNA repair pathways protect the genome against mutations but also act as an adaptive mechanism to promote drug resistance. In this review, we scrutinize the molecular mechanisms and DNA repair pathways which are conserved in trypanosomatids. The recent advances made by the genome consortiums reveal the complete genomic sequences of several pathogens. Therefore, using bioinformatics and genomic sequences, we analyze the conservation of DNA repair proteins and their key protein motifs in trypanosomatids. We thus present a comprehensive view of DNA repair processes in trypanosomatids at the crossroads of DNA repair and drug resistance.

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“…DNA binding assays revealed that it disrupted initial RAD51 binding to ssDNA, and later dsDNA binding to the RAD51-ssDNA filament 7 . A D-loop assay confirmed B02 specificity for human RAD51 over its bacterial homologue RecA and other human HR proteins 15 . In vitro, B02 inhibited irradiation-induced RAD51 foci formation, HR repair of dsDNA breaks 7 and sensitized cells to a panel of chemotherapy drugs 5,7 .…”

mentioning

confidence: 76%

“…Smaller alkanes (1-4) also displayed reduced activity. Varying spacer length in compound 17, with one methylene unit shorter (15) or longer (16), did not improve activity and indicated optimal positioning of the aromatic ring in 17. Of the substituted benzyl series, fluoro (18)(19)(20), nitro (21-22) and p-hydroxy (23) analogues showed comparable activity.…”

Section: Ar T Ic Le In F O Abstractmentioning

confidence: 97%