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Rad5 Dysregulation Drives Hyperactive Recombination at Replication Forks Resulting in Cisplatin Sensitivity and Genome Instability Eric E

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Rad5 Dysregulation Drives Hyperactive Recombination at Replication Forks Resulting in Cisplatin Sensitivity and Genome Instability Eric E 9144–9159 Nucleic Acids Research, 2019, Vol. 47, No. 17 Published online 27 July 2019 doi: 10.1093/nar/gkz631 Rad5 dysregulation drives hyperactive recombination at replication forks resulting in cisplatin sensitivity and genome instability Eric E. Bryant 1, Ivana Sunjevariˇ c´ 2, Luke Berchowitz2, Rodney Rothstein1,2,3 and Robert J.D. Reid 2,* 1Department of Biological Sciences, Columbia University, New York, NY 10027, USA, 2Department of Genetics & Development, Columbia University Irving Medical Center, New York, NY 10032, USA and 3Department of Systems Downloaded from https://academic.oup.com/nar/article/47/17/9144/5539889 by guest on 30 September 2021 Biology, Columbia University Irving Medical Center, New York, NY 10032, USA Received December 06, 2018; Revised June 14, 2019; Editorial Decision July 09, 2019; Accepted July 25, 2019 ABSTRACT cialized polymerases that can synthesize across damaged bases in a process called translesion synthesis, or through The postreplication repair gene, HLTF, is often am- bypassing lesions by continuing synthesis on undamaged plified and overexpressed in cancer. Here we model sister chromatid DNA in a process called template switch- HLTF dysregulation through the functionally con- ing (5). served Saccharomyces cerevisiae ortholog, RAD5. Translesion synthesis and template switching are some- Genetic interaction profiling and landscape enrich- times referred to as error-prone and error-free PRR re- ment analysis of RAD5 overexpression (RAD5OE)re- spectively owing to high rates of nucleotide misincorpo- veals requirements for genes involved in recombi- ration by translesion synthesis polymerases across from nation, crossover resolution, and DNA replication. damaged bases (6). However, template switching can cause While RAD5OE and rad5Δ both cause cisplatin sensi- errors through ectopic recombination, which can drive tivity and share many genetic interactions, RAD5OE loss or gain of information in repetitive DNA (7,8). Moreover, fork stalling and template switching result- specifically requires crossover resolving genes and ing in microhomology-mediated break-induced replica- drives recombination in a region of repetitive DNA. OE tion (BIR) is the proposed driving mechanism behind Remarkably, RAD5 induced recombination does disease-associated human genome rearrangements (9,10). not require other post-replication repair pathway In the absence of PRR, replication forks encountering a members, or the PCNA modification sites involved in DNA lesion are more prone to fork collapse which may regulation of this pathway. Instead, the RAD5OE phe- also result in a recombinogenic double-stranded DNA notype depends on a conserved domain necessary break and chromosomal rearrangements (11). Entry into for binding 3 DNA ends. Analysis of DNA replica- PRR is mediated by monoubiquitination of proliferat- tion intermediates supports a model in which dys- ing cell nuclear antigen (PCNA), performed in Saccha- regulated Rad5 causes aberrant template switching romyces cerevisiae by the Rad6–Rad18 complex (human / at replication forks. The direct effect of Rad5 on repli- UBE2A UBE2B-RAD18) (12). This initial modification of PCNA is thought to recruit translesion synthesis poly- cation forks in vivo, increased recombination, and merases to a stalled replication fork. Subsequent regula- cisplatin sensitivity predicts similar consequences tion of translesion synthesis/template switching pathway for dysregulated HLTF in cancer. choice is then thought to be mediated by the Mms2–Ubc13– Rad5 complex (human UBE2N-UBE2V2-HLTF), which INTRODUCTION can polyubiquitinate PCNA and perform replication fork remodeling to initiate template switching (13,14). Post-replication repair (PRR) is essential for damage Given the importance of PRR in responding to lesions tolerance during DNA replication. In the absence of caused by commonly used chemotherapeutic drugs, we this pathway, cells are acutely sensitive to intrastrand sought to determine whether this pathway incurs any fre- DNA crosslinking agents such as cisplatin––one of sev- quent genetic alterations in cancer genomes. In this study, eral platinum-based drugs used in an estimated 50% of all we perform an analysis of primary tumor samples from The chemotherapeutic regimens (1–4). PRR enables damage tol- Cancer Genome Atlas (TCGA), which reveals HLTF to be erance during replication either through recruitment of spe- *To whom correspondence should be addressed. Tel: +1 212 305 1734; Fax: +1 212 923 2090; Email: [email protected] C The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research, 2019, Vol. 47, No. 17 9145 commonly amplified and overexpressed in esophageal, uter- We find that a Rad5 ATPase mutant, defective in translo- ine and several types of squamous cell carcinoma (15). This case activity (25), only partially relieves the consequences observation corroborates previous work showing overex- of RAD5OE, whereas a Rad5 HIRAN point mutation, iden- pression of HLTF at the RNA level in several cancer cell tified using a homology model of the HLTF ssDNA bind- lines and at the protein level in hypopharyngeal squamous ing HIRAN domain, nearly eliminates the RAD5OE pheno- cell carcinomas, as well as during early stages of carcino- type. By quantitative physical analysis of DNA replication genesis in a kidney tumor model (16–18). Paradoxically, intermediates, we demonstrate that RAD5OE promotes for- HLTF is also found to be frequently silenced by promoter mation of crossover products near an early firing replica- methylation in colorectal cancers (19), leading to conflict- tion origin. These products persist in the absence of Sgs1, a ing descriptions of HLTF as both a tumor suppressor and RecQ helicase involved in crossover resolution and depend driver. Ultimately, the role of variable HLTF expression on a functional Rad5 HIRAN domain. Our data support a in carcinogenesis remains unclear. Our goal is to under- model whereby RAD5OE bypasses PRR regulatory events to stand whether dysregulation of this gene could have func- promote aberrant template switching via HIRAN domain- Downloaded from https://academic.oup.com/nar/article/47/17/9144/5539889 by guest on 30 September 2021 tional consequences for cells, especially with respect to cel- mediated replication fork remodeling. The genomic ramifi- lular fitness, response to chemotherapy and maintenance of cations of RAD5OE presented here inform the mechanistic genome stability. consequences of HLTF misexpression and its potential ef- Human HLTF and its budding yeast homolog, Rad5, are fect on carcinogenesis, genome instability, and response to remarkably conserved in domain structure and biochemical platinum-based chemotherapy. function. HLTF and Rad5 share the eponymous HIP116, Rad5p N-terminal (HIRAN) domain –– an ancient region MATERIALS AND METHODS of amino acid sequence homology that has been shown to form an OB-fold structure which can bind to the 3 end of Strains and plasmids single-stranded DNA (ssDNA) (20–23). Downstream of the All strains are RAD5 derivatives of W303 (34), unless oth- / HIRAN domain, both HLTF and Rad5 have SWI2 SNF2 erwise stated. Library strains used in our genetic screens are helicase sequence homology interrupted by a RING finger derivatives of S288C (35). Strains and plasmids constructed domain (20,24). Biochemically, both proteins have adeno- for this study, or used in validation experiments, were ver- sine triphosphate (ATP)-dependent DNA translocase ac- ified by Sanger sequencing. All genotypes for strains and tivity, and are capable of reversing model replication forks plasmids used in this study and primers used for construc- in vitro (25,26). Additionally, both proteins physically inter- tion and validation are listed in Supplementary Tables S7 act with a Rad6-Rad18 counterpart and cooperate with an and 8. Mms2-Ubc13 E2 ubiquitin ligase heterodimer to mediate Lys-63-linked PCNA polyubiquitination (12,27,28). Genet- ically, HLTF is capable of partially complementing rad5Δ in Cancer genomics ultraviolet radiation (UV)-treated yeast, albeit in a transle- Copy number and mRNA expression data for primary tu- sion synthesis deficient background28 ( ). mor samples originate from The Cancer Genome Atlas The RAD5 gene was initially discovered on the basis of (https://cancergenome.nih.gov). These data were accessed UV sensitivity, and reduced forward mutation rates in for- via the Memorial Sloan Kettering computational biology ward genetic screens performed in the late 1960s and early portal R package (http://www.cbioportal.org) which pro- 1970s and much progress has been made since in elucidat- vides access to TCGA data processed using the Broad in- ing its roles in PRR (29–32). While it is clear that Rad5 stitute GDAC firehose pipeline (https://gdac.broadinstitute. contributes to regulating PRR through PCNA modifica- org)(15,36,37). Gene level threshold GISTIC scores were tion, the mechanism of PCNA polyubiquitination in pro- used to classify the predicted ploidy of genes in a given tu- moting template switching is unclear. Furthermore, while mor sample with a score of 2 representing
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