Template Switching Research Articles

A Conserved Stem-Loop Structure within ORF5 Is a Frequent Recombination Hotspot for Porcine Reproductive and Respiratory Syndrome Virus 1 (PRRSV-1) with a Particular Modified Live Virus (MLV) Strain

The emergence of recombinant PRRSV strains has been observed for more than a decade. These recombinant viruses are characterized by a genome that contains genetic material from at least two different parental strains. Due to the advanced sequencing techniques and a growing number of data bank entries, the role of PRRSV recombinants has become increasingly important since they are sometimes associated with clinical outbreaks. Chimeric viruses observed more recently are products of PRRSV wild-type and vaccine strains. Here, we report on three PRRSV-1 isolates from geographically distant farms with differing clinical manifestations. A sequencing and recombination analysis revealed that these strains are crossovers between different wild-type strains and the same modified live virus vaccine strain. Interestingly, the recombination breakpoint of all analyzed isolates appears at the beginning of open reading frame 5 (ORF5). RNA structure predictions indicate a conserved stem loop in close proximity to the recombination hotspot, which is a plausible cause of a polymerase template switch during RNA replication. Further research into the mechanisms of the stem loop is needed to help understand the PRRSV recombination process and the role of MLVs as parental strains.

Fork-Remodeling Helicase Rad5 Preferentially Reverses Replication Forks with Gaps in the Leading Strand

DNA damage bypass pathways promote the replication of damaged DNA when replication forks stall at sites of DNA damage. Template switching is a DNA damage bypass pathway in which fork-reversal helicases convert stalled replication forks into four-way DNA junctions called chicken foot intermediates, which are subsequently extended by replicative DNA polymerases. In yeast, fork-reversal is carried out by the Rad5 helicase using an unknown mechanism. To better understand the mechanism of Rad5 and its specificity for different fork DNA substrates, we used a FRET-based assay to observe fork reversal in real time. We examined the ability of Rad5 to bind and catalyze the reversal of various fork DNA substrates in the presence of short gaps in the leading or lagging strand as well as in the presence or absence of RPA and RNA primers in the lagging strand. We found that Rad5 preferentially reverses fork DNA substrates with short gaps (10 to 30 nt.) in the leading strand. Thus, Rad5 preferentially reverses fork DNA substrates that form chicken foot intermediates with 5′ overhangs that can be extended by replicative DNA polymerases during the subsequent steps of template switching.

Rad5 participates in lesion bypass through its Rev1-binding and ubiquitin ligase domains, but not through its helicase function.

DNA Damage Tolerance (DDT) functions to bypass replication-blocking lesions and is divided into two distinct pathways: error-prone Translesion Synthesis (TLS) and error-free Damage Avoidance (DA). Rad5 is a multifunctional protein that is involved in these DDT processes. Saccharomyces cerevisiae Rad5 contains three well defined domains: a RING domain that promotes PCNA polyubiquitination, a ssDNA-dependent ATPase/helicase domain, and a Rev1-binding domain. Both the RING domain and the ATPase/helicase domain are conserved in human Rad5 ortholog HLTF. In this study we used domain-specific mutants to address the contribution of each of the Rad5 domains to the lesion tolerance. We demonstrate that the two critical functions of Rad5 during DNA damage tolerance are the activation of template switching through polyubiquitination of PCNA and the recruitment of TLS polymerases, and that loss of one of those functions can be compensated by increased usage of the other. We also show that, unlike previously suggested, the helicase activity does not play any role in lesion tolerance.

Rad52’s DNA annealing activity drives template switching associated with restarted DNA replication

It is thought that many of the simple and complex genomic rearrangements associated with congenital diseases and cancers stem from mistakes made during the restart of collapsed replication forks by recombination enzymes. It is hypothesised that this recombination-mediated restart process transitions from a relatively accurate initiation phase to a less accurate elongation phase characterised by extensive template switching between homologous, homeologous and microhomologous DNA sequences. Using an experimental system in fission yeast, where fork collapse is triggered by a site-specific replication barrier, we show that ectopic recombination, associated with the initiation of recombination-dependent replication (RDR), is driven mainly by the Rad51 recombinase, whereas template switching, during the elongation phase of RDR, relies more on DNA annealing by Rad52. This finding provides both evidence and a mechanistic basis for the transition hypothesis.

Is the Intergenic Region of Aedes aegypti Totivirus a Recombination Hotspot?

The genus totivirus in the family Totiviridae contains double-stranded RNA viruses. Their genome has two open reading frames (ORFs) that encode capsid protein (CP) and RNA-dependent RNA polymerase (RdRp). The toti-like viruses recently identified in Anopheles sp. and Aedes aegypti mosquitoes (AaTV) share the same genome organization as other totiviruses. The AaTVs that have been described in distinct geographical regions are monophyletic. In this study, we show that AaTV sequences can be grouped into at least three phylogenetic clades (named A, B, and C). Clades A and B are composed of AaTV sequences from mosquitoes collected in the Caribbean region (Guadeloupe), and clade C contains sequences from the USA. These clades may represent AaTV lineages that are locally adapted to their host populations. We also identified three recombinant AaTV strains circulating in mosquitoes in Guadeloupe. Although these strains have different chimeric patterns, the position of the recombination breakpoint was identical in all strains. Interestingly, this breakpoint is located in a hairpin-like structure in the intergenic region of the AaTV genome. This RNA structure may stall RNA polymerase processivity and consequently induce template switching. In vitro studies should be conducted to further investigate the biological significance of AaTV's intergenic region as a recombination hotspot.

Leveraging the replication stress response to optimize cancer therapy.

High-fidelity DNA replication is critical for the faithful transmission of genetic information to daughter cells. Following genotoxic stress, specialized DNA damage tolerance pathways are activated to ensure replication fork progression. These pathways include translesion DNA synthesis, template switching and repriming. In this Review, we describe how DNA damage tolerance pathways impact genome stability, their connection with tumorigenesis and their effects on cancer therapy response. We discuss recent findings that single-strand DNA gap accumulation impacts chemoresponse and explore a growing body of evidence that suggests that different DNA damage tolerance factors, including translesion synthesis polymerases, template switching proteins and enzymes affecting single-stranded DNA gaps, represent useful cancer targets. We further outline how the consequences of DNA damage tolerance mechanisms could inform the discovery of new biomarkers to refine cancer therapies.

Click Chemistry Enables Rapid Amplification of Full-Length Reverse Transcripts for Long-Read Third Generation Sequencing.

Here we describe the development of a novel click chemistry-based method for the generation and amplification of full-length cDNA libraries from total RNA, while avoiding the need for problematic template-switching (TS) reactions. Compared with prior efforts, our method involves neither random priming nor stochastic cDNA termination, thus enabling amplification of transcripts that were previously inaccessible via related click chemistry-based RNA sequencing techniques. A key modification involving the use of PCR primers containing two overhanging 3'-nucleotides substantially improved the read-through compatibility of the 1,4-disubstituted 1,2,3-triazole-containing cDNA, where such modifications typically hinder amplification. This allowed us to more than double the possible insert size compared with the state-of-the art click chemistry-based technique, PAC-seq. Furthermore, our method performed on par with a commercially available PCR-cDNA RNA sequencing kit, as determined by Oxford Nanopore sequencing. Given the known advantages of PAC-seq, namely, suppression of PCR artifacts, we anticipate that our contribution could enable diverse applications including improved analyses of mRNA splicing variants and fusion transcripts.

Hrq1/RECQL4 regulation is critical for preventing aberrant recombination during DNA intrastrand crosslink repair and is upregulated in breast cancer.

Human RECQL4 is a member of the RecQ family of DNA helicases and functions during DNA replication and repair. RECQL4 mutations are associated with developmental defects and cancer. Although RECQL4 mutations lead to disease, RECQL4 overexpression is also observed in cancer, including breast and prostate. Thus, tight regulation of RECQL4 protein levels is crucial for genome stability. Because mammalian RECQL4 is essential, how cells regulate RECQL4 protein levels is largely unknown. Utilizing budding yeast, we investigated the RECQL4 homolog, HRQ1, during DNA crosslink repair. We find that Hrq1 functions in the error-free template switching pathway to mediate DNA intrastrand crosslink repair. Although Hrq1 mediates repair of cisplatin-induced lesions, it is paradoxically degraded by the proteasome following cisplatin treatment. By identifying the targeted lysine residues, we show that preventing Hrq1 degradation results in increased recombination and mutagenesis. Like yeast, human RECQL4 is similarly degraded upon exposure to crosslinking agents. Furthermore, over-expression of RECQL4 results in increased RAD51 foci, which is dependent on its helicase activity. Using bioinformatic analysis, we observe that RECQL4 overexpression correlates with increased recombination and mutations. Overall, our study uncovers a role for Hrq1/RECQL4 in DNA intrastrand crosslink repair and provides further insight how misregulation of RECQL4 can promote genomic instability, a cancer hallmark.

Chimeric DNA byproducts in strand displacement amplification using the T7 replisome.

Recent advances in next generation sequencing technologies enable reading DNA molecules hundreds of kilobases in length and motivate development of DNA amplification methods capable of producing long amplicons. In vivo, DNA replication is performed not by a single polymerase enzyme, but multiprotein complexes called replisomes. Here, we investigate strand-displacement amplification reactions using the T7 replisome, a macromolecular complex of a helicase, a single-stranded DNA binding protein, and a DNA polymerase. The T7 replisome may initiate processive DNA synthesis from DNA nicks, and the reaction of a 48 kilobase linear double stranded DNA substrate with the T7 replisome and nicking endonucleases is shown to produce discrete DNA amplicons. To gain a mechanistic understanding of this reaction, we utilized Oxford Nanopore long-read sequencing technology. Sequence analysis of the amplicons revealed chimeric DNA reads and uncovered a connection between template switching and polymerase exonuclease activity. Nanopore sequencing provides insight to guide the further development of isothermal amplification methods for long DNA, and our results highlight the need for high-specificity, high-turnover nicking endonucleases to initiate DNA amplification without thermal denaturation.

On the Origins of Omicron's Unique Spike Gene Insertion.

The emergence of a heavily mutated SARS-CoV-2 variant (Omicron; Pango lineage B.1.1.529 and BA sublineages) and its rapid spread to over 75 countries raised a global public health alarm. Characterizing the mutational profile of Omicron is necessary to interpret its clinical phenotypes which are shared with or distinctive from those of other SARS-CoV-2 variants. We compared the mutations of the initially circulating Omicron variant (now known as BA.1) with prior variants of concern (Alpha, Beta, Gamma, and Delta), variants of interest (Lambda, Mu, Eta, Iota, and Kappa), and ~1500 SARS-CoV-2 lineages constituting ~5.8 million SARS-CoV-2 genomes. Omicron’s Spike protein harbors 26 amino acid mutations (23 substitutions, 2 deletions, and 1 insertion) that are distinct compared to other variants of concern. While the substitution and deletion mutations appeared in previous SARS-CoV-2 lineages, the insertion mutation (ins214EPE) was not previously observed in any other SARS-CoV-2 lineage. Here, we consider and discuss various mechanisms through which the nucleotide sequence encoding for ins214EPE could have been acquired, including local duplication, polymerase slippage, and template switching. Although we are not able to definitively determine the mechanism, we highlight the plausibility of template switching. Analysis of the homology of the inserted nucleotide sequence and flanking regions suggests that this template-switching event could have involved the genomes of SARS-CoV-2 variants (e.g., the B.1.1 strain), other human coronaviruses that infect the same host cells as SARS-CoV-2 (e.g., HCoV-OC43 or HCoV-229E), or a human transcript expressed in a host cell that was infected by the Omicron precursor.

Melatonin: Regulation of Viral Phase Separation and Epitranscriptomics in Post-Acute Sequelae of COVID-19.

The relentless, protracted evolution of the SARS-CoV-2 virus imposes tremendous pressure on herd immunity and demands versatile adaptations by the human host genome to counter transcriptomic and epitranscriptomic alterations associated with a wide range of short- and long-term manifestations during acute infection and post-acute recovery, respectively. To promote viral replication during active infection and viral persistence, the SARS-CoV-2 envelope protein regulates host cell microenvironment including pH and ion concentrations to maintain a high oxidative environment that supports template switching, causing extensive mitochondrial damage and activation of pro-inflammatory cytokine signaling cascades. Oxidative stress and mitochondrial distress induce dynamic changes to both the host and viral RNA m6A methylome, and can trigger the derepression of long interspersed nuclear element 1 (LINE1), resulting in global hypomethylation, epigenetic changes, and genomic instability. The timely application of melatonin during early infection enhances host innate antiviral immune responses by preventing the formation of “viral factories” by nucleocapsid liquid-liquid phase separation that effectively blockades viral genome transcription and packaging, the disassembly of stress granules, and the sequestration of DEAD-box RNA helicases, including DDX3X, vital to immune signaling. Melatonin prevents membrane depolarization and protects cristae morphology to suppress glycolysis via antioxidant-dependent and -independent mechanisms. By restraining the derepression of LINE1 via multifaceted strategies, and maintaining the balance in m6A RNA modifications, melatonin could be the quintessential ancient molecule that significantly influences the outcome of the constant struggle between virus and host to gain transcriptomic and epitranscriptomic dominance over the host genome during acute infection and PASC.

A Bayesian approach to infer recombination patterns in coronaviruses

As shown during the SARS-CoV-2 pandemic, phylogenetic and phylodynamic methods are essential tools to study the spread and evolution of pathogens. One of the central assumptions of these methods is that the shared history of pathogens isolated from different hosts can be described by a branching phylogenetic tree. Recombination breaks this assumption. This makes it problematic to apply phylogenetic methods to study recombining pathogens, including, for example, coronaviruses. Here, we introduce a Markov chain Monte Carlo approach that allows inference of recombination networks from genetic sequence data under a template switching model of recombination. Using this method, we first show that recombination is extremely common in the evolutionary history of SARS-like coronaviruses. We then show how recombination rates across the genome of the human seasonal coronaviruses 229E, OC43 and NL63 vary with rates of adaptation. This suggests that recombination could be beneficial to fitness of human seasonal coronaviruses. Additionally, this work sets the stage for Bayesian phylogenetic tracking of the spread and evolution of SARS-CoV-2 in the future, even as recombinant viruses become prevalent.

Thousands of human mutation clusters are explained by short-range template switching.

Variation within human genomes is unevenly distributed, and variants show spatial clustering. DNA replication-related template switching is a poorly known mutational mechanism capable of causing major chromosomal rearrangements as well as creating short inverted sequence copies that appear as local mutation clusters in sequence comparisons. In this study, haplotype-resolved genome assemblies representing 25 human populations and multinucleotide variants aggregated from 140,000 human sequencing experiments were reanalyzed. Local template switching could explain thousands of complex mutation clusters across the human genome, the loci segregating within and between populations. During the study, computational tools were developed for identification of template switch events using both short-read sequencing data and genotype data, and for genotyping candidate loci using short-read data. The characteristics of template-switch mutations complicate their detection, and widely used analysis pipelines for short-read sequencing data, normally capable of identifying single nucleotide changes, were found to miss template-switch mutations of tens of base pairs, potentially invalidating medical genetic studies searching for a causative allele behind genetic diseases. Combined with the massive sequencing data now available for humans, the novel tools described here enable building catalogs of affected loci and studying the cellular mechanisms behind template switching in both healthy organisms and disease.

Abstract LB545: Structure, dynamics and consequences of replication stress-induced structural variants in hepatocellular carcinoma

Abstract Oncogene activation leads to replication stress and promotes genomic instability. Here we combine optical mapping and whole genome sequencing to explore in-depth the nature of structural variants (SVs) induced by replication stress in cyclin-activated hepatocellular carcinomas (CCN-HCC). In addition to classical tandem duplications, CCN-HCC display frequent intra- and inter-chromosomal templated insertion cycles (TIC) likely resulting from template switching events. Template switching preferentially involves active topologically associated domains that are close in the 3D genome organization. Template sizes depend on the type of cyclin activation and are coordinated within each TIC. Replication stress induces continuous accumulation of SVs during CCN-HCC progression, fostering the acquisition of new driver alterations and large-scale copy-number changes at TIC borders. Together, this analysis sheds light on the mechanisms, dynamics and consequences of SV accumulation in tumors with oncogene-induced replication stress. Citation Format: Quentin Bayard, Pierre Cordier, Camille Péneau, Sandrine Imbeaud, Theo Z. Hirsch, Victor Renault, Jean-Charles Nault, Jean-Frédéric Blanc, Julien Calderaro, Chantal Desdouets, Jessica Zucman-Rossi, Eric Letouzé. Structure, dynamics and consequences of replication stress-induced structural variants in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB545.

Post-Translational Modifications of PCNA: Guiding for the Best DNA Damage Tolerance Choice.

The sliding clamp PCNA is a multifunctional homotrimer mainly linked to DNA replication. During this process, cells must ensure an accurate and complete genome replication when constantly challenged by the presence of DNA lesions. Post-translational modifications of PCNA play a crucial role in channeling DNA damage tolerance (DDT) and repair mechanisms to bypass unrepaired lesions and promote optimal fork replication restart. PCNA ubiquitination processes trigger the following two main DDT sub-pathways: Rad6/Rad18-dependent PCNA monoubiquitination and Ubc13-Mms2/Rad5-mediated PCNA polyubiquitination, promoting error-prone translation synthesis (TLS) or error-free template switch (TS) pathways, respectively. However, the fork protection mechanism leading to TS during fork reversal is still poorly understood. In contrast, PCNA sumoylation impedes the homologous recombination (HR)-mediated salvage recombination (SR) repair pathway. Focusing on Saccharomyces cerevisiae budding yeast, we summarized PCNA related-DDT and repair mechanisms that coordinately sustain genome stability and cell survival. In addition, we compared PCNA sequences from various fungal pathogens, considering recent advances in structural features. Importantly, the identification of PCNA epitopes may lead to potential fungal targets for antifungal drug development.

DNA Sequence Specificity Reveals a Role of the HLTF HIRAN Domain in the Recognition of Trinucleotide Repeats.

DNA damage tolerance (DDT) pathways enable cells to cope with a variety of replication blocks that threaten their ability to complete DNA replication. Helicase-like transcription factor (HLTF) plays a central role in the error-free DDT pathway, template switching (TS), by serving as a ubiquitin ligase to polyubiquitinate the DNA sliding clamp PCNA, which promotes TS initiation. HLTF also serves as an ATP-dependent DNA translocase facilitating replication fork remodeling. The HIP116, Rad5p N-terminal (HIRAN) domain of HLTF specifically recognizes the unmodified 3'-end of single-stranded DNA (ssDNA) at stalled replication forks to promote fork regression. Several crystal structures of the HIRAN domain in complex with ssDNA have been reported; however, optimal ssDNA sequences for high-affinity binding with the domain have not been described. Here we elucidated DNA sequence preferences of HLTF HIRAN through systematic studies of its binding to ssDNA substrates using fluorescence polarization assays and a computational analysis of the ssDNA:HIRAN interaction. These studies reveal that the HLTF HIRAN domain preferentially recognizes a (T/C)TG sequence at the 3'-hydroxyl ssDNA end, which occurs in the CTG trinucleotide repeat (TNR) regions that are susceptible to expansion and deletion mutations identified in neuromuscular and neurodegenerative disorders. These findings support a role for HLTF in maintaining the stability of difficult to replicate TNR microsatellite regions.

2 5’ Untranslated Region-Based Detection of Genetically Diverse APPV Strains in US Midwest

Abstract Atypical porcine pestivirus (APPV), an RNA virus member of the Flaviviridae family, has been associated with congenital tremor in newborn piglets. Previous qPCR-based assays, targeting the polyprotein coding sequences of APPV, were unable to detect the virus in novel cases of congenital tremor originated from multiple swine farms from the U.S. Midwest (MW). The polyprotein coding genes of APPV were characterized by a low level of sequence conservation ranging from 58.2 to 70.7% among 15 global APPV strains. In contrast, the 5’ untranslated region (UTR) was found to have a much greater degree of sequence conservation of 81.0% seen among the 15 global and new 4 MW strains, and as high as 91.5% when comparing only 5 MW strains. The complete 5’ UTR sequence of the APPV strains originated from MW was obtained by utilizing the template switching approach followed by amplification and dideoxy sequencing. Notably, previously identified potentially crucial regions for the correct function of the APPV internal ribosome entry site (IRES) were found to overlap with the most highly conserved 5’UTR segments, suggesting their functional role in viral protein translation. By targeting 100% conserved 5’UTR regions across 19 global and MW strains, a new qPCR assay was designed which was able to detect APPV in well documented cases of congenital tremor which originated from 5 MW farm sites (1-18 samples/site). The assay was found to have a detection sensitivity of 6.5 APPV copies (Cq ~38.4). Due to the potential functional importance of the highly conserved 5’UTR sequences targeted by our newly developed qPCR assay, we expect that assays targeting this region would broadly detect APPV strains that are diverse in space and time.

EP402: Partial trisomy of chromosome 22 mediated by chromoanasynthesis in an 8-month-old male

Chromoanasynthesis is a subtype of chromoanagenesis, a term used to describe the single-step processes by which complex chromosomal rearrangements lead to localized structural genomic reorganization of one or a few chromosomes. During chromoanasynthesis, complex chromosomal rearrangements are mediated by replication-based mechanisms including fork stalling and template switching (FoSTeS) or by microhomology-mediated break-induced replication (MMBIR). As a result, highly reorganized chromosomes with multiple copy number changes including deletions, duplications, and triplications are generated.

Abstract P2-06-04: Pathognomonic long molecule footprints of backup repair pathways in homologous recombination deficient cancers

Abstract Background: Cancer genomes provide a durable record of the genetic alterations that are acquired during normal cell development and carcinogenesis from DNA damage and DNA repair defects. As DNA repair-deficient tumors often become dependent on backup repair pathways, mutational signatures found in such tumors are thought to reflect the absence of a particular repair pathway as well as the activity of the backup repair mechanism responsible for maintaining genome integrity. While homologous recombination (HR) deficiency is primarily a disorder of double-strand break (DSB) repair, the mutation classes most specifically associated with HR-deficiency in cancer are paradoxically small variants, namely single nucleotide variants and short deletions. Mechanistic and cytogenetic studies, however, indicate that HR-deficiency should compromise structural genomic integrity and yield complex rearrangements. Here we elucidate complex structural variants that are specific for HR-deficient cancers and identify rearrangements that differentiate BRCA1 from BRCA2 loss and illuminate divergent backup DNA repair mechanisms. Methods: To investigate the role of complex SVs in HR-deficient cancers, we assembled a cohort of 2,367 WGS profiles from four tumor types (breast, ovarian, prostate, and pancreatic cancer) known to be associated with HR-deficiency. We identified 48 BRCA1-/- and 87 BRCA2-/- cases and called samples lacking (mono- or biallelic) mutations in BRCA1, BRCA2, or any other HR associated gene (e.g. PALB2, RAD51C) as wild-type. To validate observed structural variant patterns associated with homologous recombination deficiency from this larger dataset, a new cohort of 49 cases of invasive breast cancer with known BRCA1 (N = 29) or BRCA2 (N = 20) deficiency was collected as part of a prospective research study at Memorial Sloan Kettering Cancer Center and sequenced with 10X-linked read WGS in addition to standard Illumina short-read WGS. Results: Analysis of nearly 2,400 short-read whole genomes revealed distinct quasi-reciprocal structural variants (SVs) highly enriched in BRCA1-/- versus BRCA2-/- cancers. Applying high physical coverage (>150X) long molecule WGS to 49 tumor-normal pairs from breast cancer patients with inherited BRCA1 or BRCA2 loss-of-function mutations, we show that these SVs are associated with distinct cis or trans somatic allelic phases despite having nearly identical short read WGS footprints. Trans (crossover) outcomes were found to give rise to large-scale chromosomal variants that mediate the loss-of-heterozygosity patterns previously described as a cardinal feature of HR-deficient cancers. We find that these quasi-reciprocal SVs can be explained as distinct template switching outcomes from a shared intermediate arising after replication fork stalling in HR-deficient cancers. Furthermore, our WGS analyses reveal that BRCA2 genomes are highly enriched in deletions that harbor long (50-1000bp) tracts of inexact microhomology. These events, validated by long molecule WGS, indicate that single-stranded annealing serves as an active backup repair pathway in BRCA2-/- but not BRCA1-/- cancers. Conclusions: These results provide direct genomic evidence linking large-scale structural changes in HR-deficient tumors with specific backup repair pathways that suggest novel, therapeutically targetable dependencies. Our findings elucidate backup repair mechanisms responsible for generating structural variation in HR-deficient tumors, demonstrate the genotype-specific divergence of such compensatory DNA repair, and provide genomic features that improve the detection accuracy of HR-deficiency with utility for the optimal selection of treatment. Citation Format: Jeremy Setton, Kevin Hadi, Huasong Tian, Arnaud Da Cruz Paula, Joel Rosiene, Zi-Ning Choo, Julie Behr, Xiaotong Yao, Olivier Elemento, Britta Weigelt, Nadeem Riaz, Jorge S Reis-Filho, Marcin Imielinski, Simon N Powell. Pathognomonic long molecule footprints of backup repair pathways in homologous recombination deficient cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-06-04.

DNA Damage Tolerance Pathways in Human Cells: A Potential Therapeutic Target.

DNA lesions arising from both exogenous and endogenous sources occur frequently in DNA. During DNA replication, the presence of unrepaired DNA damage in the template can arrest replication fork progression, leading to fork collapse, double-strand break formation, and to genome instability. To facilitate completion of replication and prevent the generation of strand breaks, DNA damage tolerance (DDT) pathways play a key role in allowing replication to proceed in the presence of lesions in the template. The two main DDT pathways are translesion synthesis (TLS), which involves the recruitment of specialized TLS polymerases to the site of replication arrest to bypass lesions, and homology-directed damage tolerance, which includes the template switching and fork reversal pathways. With some exceptions, lesion bypass by TLS polymerases is a source of mutagenesis, potentially contributing to the development of cancer. The capacity of TLS polymerases to bypass replication-blocking lesions induced by anti-cancer drugs such as cisplatin can also contribute to tumor chemoresistance. On the other hand, during homology-directed DDT the nascent sister strand is transiently utilised as a template for replication, allowing for error-free lesion bypass. Given the role of DNA damage tolerance pathways in replication, mutagenesis and chemoresistance, a more complete understanding of these pathways can provide avenues for therapeutic exploitation. A number of small molecule inhibitors of TLS polymerase activity have been identified that show synergy with conventional chemotherapeutic agents in killing cancer cells. In this review, we will summarize the major DDT pathways, explore the relationship between damage tolerance and carcinogenesis, and discuss the potential of targeting TLS polymerases as a therapeutic approach.