EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart

Hambarde, Shashank; Tsai, Chi Lin; Pandita, Raj K.; Bacolla, Albino; Maitra, Anirban; Charaka, Vijay; Hunt, Clayton R.; Kumar, Rakesh; Limbo, Oliver; Meur, Rémy Le; Chazin, Walter J.; Tsutakawa, Susan E.; Russell, Paul; Schlacher, Katharina; Pandita, Tej K.; Tainer, John A.

doi:10.1016/j.molcel.2021.05.027

Cited by 31 publications

(24 citation statements)

References 120 publications

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“…During replication stress, stalled replication forks are processed by helicases and nucleases for repair and restart, and RPA binds ssDNA to recruit ATR kinase for regulation of stress response by activating fork remodelers, protectors, and restart complexes to ensure genome integrity ( Saldivar et al, 2017 ). Stalled forks can be reversed by fork remodelers (SMARCAL1, HLTF, and ZRANB3), stabilized by fork protection factors (BRCA1/2, PARP1, and Fanconi anemia proteins), and restarted by helicase–nuclease complexes (BLM-EXO5, WRN-DNA2) to maintain genome stability ( Thangavel et al, 2015 ; Liao et al, 2018 ; Hambarde et al, 2021 ). To prevent unscheduled nuclease degradation, efficient stalled replication fork restart by BLM-EXO5-RPA complex is a critical step to resume replication.…”

Section: Introductionmentioning

confidence: 99%

“…To prevent unscheduled nuclease degradation, efficient stalled replication fork restart by BLM-EXO5-RPA complex is a critical step to resume replication. Defects in BLM-EXO5 complex decrease replication fork restart frequency and result in increased frequencies of chromosomal radials and sister-chromatid exchange, characteristics of Bloom syndrome (BS) patients, who are susceptible to cancer ( Cunniff et al, 2017 ; Hambarde et al, 2021 ). Furthermore, BLM-deficient cells increase genetic exchanges between homologous chromosomes, resulting in loss of heterozygosity (LOH), which typically increases in cancer and aging cells.…”

Section: Introductionmentioning

confidence: 99%

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Decoding Cancer Variants of Unknown Significance for Helicase–Nuclease–RPA Complexes Orchestrating DNA Repair During Transcription and Replication

Tsutakawa

Bacolla

Katsonis³

et al. 2021

Front. Mol. Biosci.

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All tumors have DNA mutations, and a predictive understanding of those mutations could inform clinical treatments. However, 40% of the mutations are variants of unknown significance (VUS), with the challenge being to objectively predict whether a VUS is pathogenic and supports the tumor or whether it is benign. To objectively decode VUS, we mapped cancer sequence data and evolutionary trace (ET) scores onto crystallography and cryo-electron microscopy structures with variant impacts quantitated by evolutionary action (EA) measures. As tumors depend on helicases and nucleases to deal with transcription/replication stress, we targeted helicase–nuclease–RPA complexes: (1) XPB-XPD (within TFIIH), XPF-ERCC1, XPG, and RPA for transcription and nucleotide excision repair pathways and (2) BLM, EXO5, and RPA plus DNA2 for stalled replication fork restart. As validation, EA scoring predicts severe effects for most disease mutations, but disease mutants with low ET scores not only are likely destabilizing but also disrupt sophisticated allosteric mechanisms. For sites of disease mutations and VUS predicted to be severe, we found strong co-localization to ordered regions. Rare discrepancies highlighted the different survival requirements between disease and tumor mutations, as well as the value of examining proteins within complexes. In a genome-wide analysis of 33 cancer types, we found correlation between the number of mutations in each tumor and which pathways or functional processes in which the mutations occur, revealing different mutagenic routes to tumorigenesis. We also found upregulation of ancient genes including BLM, which supports a non-random and concerted cancer process: reversion to a unicellular, proliferation-uncontrolled, status by breaking multicellular constraints on cell division. Together, these genes and global analyses challenge the binary “driver” and “passenger” mutation paradigm, support a gradient impact as revealed by EA scoring from moderate to severe at a single gene level, and indicate reduced regulation as well as activity. The objective quantitative assessment of VUS scoring and gene overexpression in the context of functional interactions and pathways provides insights for biology, oncology, and precision medicine.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Decoding Cancer Variants of Unknown Significance for Helicase–Nuclease–RPA Complexes Orchestrating DNA Repair During Transcription and Replication

Tsutakawa

Bacolla

Katsonis³

et al. 2021

Front. Mol. Biosci.

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“…For example, during transcription, the pretargeting of base excision-repair complexes to open chromatin coincides with local variations in mutation rates across the genome resulting from differential repair of oxidized DNA base damage ( 49 ). Likewise, during replication, cells have multiple protective mechanisms in place to safeguard the genome against replicative stress, including ATR-mediated stalled replication-fork restart by the EXO5–BLM complex ( 50 ). Notably, the study of CFSs, regions of the genome that are inherently vulnerable to replication stress, has illuminated multiple proteins involved in these mechanisms.…”

Section: Discussionmentioning

confidence: 99%

Translesion polymerase eta both facilitates DNA replication and promotes increased human genetic variation at common fragile sites

Twayana

Bacolla

Barreto-Galvez

et al. 2021

Proc. Natl. Acad. Sci. U.S.A.

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Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. They are hotspots for chromosomal instability in cancer. Repetitive sequences located at CFS loci are inefficiently copied by replicative DNA polymerase (Pol) delta. However, translesion synthesis Pol eta has been shown to efficiently polymerize CFS-associated repetitive sequences in vitro and facilitate CFS stability by a mechanism that is not fully understood. Here, by locus-specific, single-molecule replication analysis, we identified a crucial role for Pol eta (encoded by the gene POLH) in the in vivo replication of CFSs, even without exogenous stress. We find that Pol eta deficiency induces replication pausing, increases initiation events, and alters the direction of replication-fork progression at CFS-FRA16D in both lymphoblasts and fibroblasts. Furthermore, certain replication pause sites at CFS-FRA16D were associated with the presence of non-B DNA-forming motifs, implying that non-B DNA structures could increase replication hindrance in the absence of Pol eta. Further, in Pol eta-deficient fibroblasts, there was an increase in fork pausing at fibroblast-specific CFSs. Importantly, while not all pause sites were associated with non-B DNA structures, they were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. From these findings, we propose that Pol eta replicating through CFSs may result in genetic variations found in the human population at these sites.

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“…The RecQ–like BLM helicase partners with EXO5 and EEPD1 nucleases for stalled DNA replication restart and maintenance of genome integrity ( 62 , 63 ). BLM deficiency in Bloom syndrome (BS) causes increased expression of inflammatory genes through the cGAS–STING–IRF3 pathway, suggesting it prevents unchecked inflammatory gene responses ( 64 ).…”

Section: Ddr In Innate Immunitymentioning

confidence: 99%

“…8-OHG stabilizes DNA against degradation by the cytosolic DNA exonuclease TREX1, leading to accumulated cytosolic DNA and increased cGAS activation ( 70 ). This ROS effect can be amplified by vicious cycles of oxidative damage and iron release from ROS-sensitive 4Fe-4S co-factors in multiple replication and repair proteins ( 62 , 71 – 73 ).…”

Section: Ddr In Innate Immunitymentioning

confidence: 99%

Function and Molecular Mechanism of the DNA Damage Response in Immunity and Cancer Immunotherapy

Yin

Lees‐Miller

et al. 2021

Front. Immunol.

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The DNA damage response (DDR) is an organized network of multiple interwoven components evolved to repair damaged DNA and maintain genome fidelity. Conceptually the DDR includes damage sensors, transducer kinases, and effectors to maintain genomic stability and accurate transmission of genetic information. We have recently gained a substantially improved molecular and mechanistic understanding of how DDR components are interconnected to inflammatory and immune responses to stress. DDR shapes both innate and adaptive immune pathways: (i) in the context of innate immunity, DDR components mainly enhance cytosolic DNA sensing and its downstream STimulator of INterferon Genes (STING)-dependent signaling; (ii) in the context of adaptive immunity, the DDR is needed for the assembly and diversification of antigen receptor genes that is requisite for T and B lymphocyte development. Imbalances between DNA damage and repair impair tissue homeostasis and lead to replication and transcription stress, mutation accumulation, and even cell death. These impacts from DDR defects can then drive tumorigenesis, secretion of inflammatory cytokines, and aberrant immune responses. Yet, DDR deficiency or inhibition can also directly enhance innate immune responses. Furthermore, DDR defects plus the higher mutation load in tumor cells synergistically produce primarily tumor-specific neoantigens, which are powerfully targeted in cancer immunotherapy by employing immune checkpoint inhibitors to amplify immune responses. Thus, elucidating DDR-immune response interplay may provide critical connections for harnessing immunomodulatory effects plus targeted inhibition to improve efficacy of radiation and chemotherapies, of immune checkpoint blockade, and of combined therapeutic strategies.

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EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart

Cited by 31 publications

References 120 publications

Decoding Cancer Variants of Unknown Significance for Helicase–Nuclease–RPA Complexes Orchestrating DNA Repair During Transcription and Replication

Decoding Cancer Variants of Unknown Significance for Helicase–Nuclease–RPA Complexes Orchestrating DNA Repair During Transcription and Replication

Translesion polymerase eta both facilitates DNA replication and promotes increased human genetic variation at common fragile sites

Function and Molecular Mechanism of the DNA Damage Response in Immunity and Cancer Immunotherapy

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