SUMO orchestrates multiple alternative DNA-protein crosslink repair pathways
Posted on: 24 July 2020
Preprint posted on 10 June 2020
Article now published in Cell Reports at http://dx.doi.org/10.1016/j.celrep.2021.110034
SUMO promotes DNA-protein crosslink (DPC) repair - until it doesn’t! Cell cycle-dependent SUMO conjugates regulate DPC repair pathway choice.
Selected by Vanessa KellnerCategories: cell biology, genetics, molecular biology
Background:
The frequent physical interaction between DNA and various proteins is an integral aspect of both the duplication and the translation of genetic information in all kingdoms of life. If one such transient interaction becomes stabilized either through exogenous chemicals or abortive reactions, the resulting covalent DNA-protein crosslink (DPC) needs to be repaired by dedicated mechanisms to prevent genome instability.
A well-studied example of a DPC is Topoisomerase 1 (Top1), an enzyme that relieves DNA supercoiling by transiently nicking the DNA, thereby creating a covalently attached reaction intermediate. If the reaction cannot be completed (e.g. in the presence of the Top1 inhibitor CPT), the resulting Top1 cleavage complex (Top1cc) needs to be processed and repaired.
Repair of Top1cc requires partial degradation of the bulky protein followed by processing of the DNA to remove any traces of the crosslinked protein. Tyrosyl-DNA phosphodiesterase 1 (Tdp1) can directly hydrolyze the bond between Top1 and DNA and does so most likely after initial proteolytic digest of Top1. Partial proteolysis can be carried out by the metalloprotease Wss1 (mammalian SPRTN) or the aspartic protease Ddi1, as well as the canonical 26S proteasome machinery. Following degradation, DNA repair pathways like nucleotide or base excision repair, or several nucleases fully repair the remaining DNA damage.
Posttranslational modifications such as phosphorylation, ubiquitylation, and sumoylation are implicated in regulating the pathway choice for DPC repair, but their exact function remains incompletely understood. For example, SUMO is conjugated to both Top1 itself and several DNA repair factors in yeast and mammals. While some studies show more efficient processing of Top1ccs upon sumoylation, others report an inhibitory effect of SUMO conjugates.
In the preprint highlighted here, the authors investigate this discrepancy and show that SUMO conjugation does indeed play a dual role in DPC repair and is regulated by the cell cycle. The work is based on a genetic interaction screen that the authors previously published (Serbyn et al, 2020).
SAturated Transposon Analysis in Yeast (SATAY) is an unbiased screen for both positive and negative genetic interactions in yeast (Michel et al, 2017). It relies on the excision and subsequent random genomic insertion of a plasmid-borne transposon followed by high-throughput sequencing of a large pool of transposon mutants. In this pool, transposon-free regions in the genome reveal essential genes (i.e. insertion of the transposon kills the cell), while overrepresented areas of insertion might show suppressive and beneficial effects. Using this approach in cells lacking both Tdp1 and Wss1, the authors were able to identify the Ddi1 protease as an important backup for Wss1 and the 26S proteasome in the removal of DPCs.
Key findings:
Components of the SUMO pathway are toxic in cells lacking both Tdp1 and Wss1
The previously published SATAY screen (Serbyn et al, 2020) identified SUMO pathway components as genetic suppressors of the growth defect observed for cells defective for Tdp1 and Wss1. This finding held true in validation experiments: A reduction of activity or complete loss of the SUMO ligase Siz2 rescued the severe growth defect and CPT sensitivity of Tdp1- and Wss1-deficient cells and led to a more efficient repair of Top1ccs.
Sumoylation of factors other than Top1 contributes to DPC repair
Surprisingly, the sumoylation target of Siz2 was not Top1 itself, and mutation of the known Top1 sumoylation sites did not rescue growth or CPT sensitivity of cells lacking Tdp1 and Wss1. Stable Isotope Labeling with Amino acids in Cell culture and mass spectrometry (SILAC-MS) was unable to identify strong candidate targets for Siz2-dependent sumoylation and the few DNA repair factors that did show reduced levels of Siz2-dependent sumoylation in the SILAC-MS data did not affect growth and CPT sensitivity of Tdp1- and Wss1-defective cells.
Local SUMO levels at the DPC site modulate DPC repair in a cell-cycle dependent manner
A well-studied site-specific DPC model system (the Flp-nick system) gave valuable insight into the dual function of SUMO at DPCs: ChIP-Seq experiments showed that tdp1Δ wss1Δ mutants accumulated Siz2-dependent SUMO in the vicinity of the DPC. While cells were in G1 phase of the cell cycle, sumoylation promoted repair of the DPC followed by removal of SUMO conjugates. But if sumoylation persisted into S phase in the absence of Tdp1 and Wss1, it delayed efficient repair.
Alternative pathways of Top1cc repair in cells lacking Tdp1, Wss1, and Siz2
Synthetic lethal interactions uncovered in a SATAY screen using tdp1Δ wss1Δ siz2Δ cells revealed potential alternative pathways that are activated upon loss of sumoylation at DPCs. Among those hits were several factors previously implicated in DPC repair: structure-specific endonucleases (Rad27, Rad1), DNA helicases (Srs2, RecQ helicase complex), the DNA damage checkpoint (9-1-1 complex, Rad9), and the Ddi1 protease.
The Model: SUMO has a dual role in the temporal and (potentially) spatial regulation of DPC repair
Initial sumoylation of Top1cc and its vicinity helps to recruit Wss1 and its partner Cdc48 to the site of damage to promote proteolysis. Sumoylation also recruits SUMO-dependent ubiquitin ligases that attach ubiquitin (Ub) chains as well as mixed Ub-SUMO chains to Top1 and other proteins to further promote degradation of Top1. One or several different nucleases then eliminate Top1 from DNA. Excess sumoylation that persists into S phase (in the absence of Wss1 or upon very high levels of Top1ccs) could block alternative replication-dependent proteases such as Ddi1 from accessing the DPC, thereby delaying efficient repair in S phase. SUMO conjugation to DPC sites thus controls temporal separation of competing repair mechanisms. But it might also exert spatial control of DPC repair: Some types of damage, such as DNA double stranded breaks, are known to localize to the nuclear periphery. Bound to nuclear pore complexes, the SUMO protease Ulp1 removes SUMO from proteins and could thus present a mechanism that restricts sumoylation and counteracts persistence of SUMO conjugates into S phase.
What I like about this work:
DNA-protein crosslinks present a severe obstacle for DNA replication, and different pathways are known to repair them. The presence of such redundancy and the strong need for faithful repair suggests that the pathway choice must be tightly regulated. SUMO conjugation has been previously reported to play both a promoting and an inhibitory role, but it was not clear in which situation either of those roles would be prominent. In this preprint, the authors present a plausible scenario for how SUMO can act either way. I am certain that further work based on this preprint will help to unravel the fine details of DPC repair. This will be especially important to help understand how acquired resistance to cancer drugs that cause DPCs (such as camptothecin or etoposide) can be overcome.
Questions to the authors:
- In your Flp-nick experiments, you perform ChIP-qPCR for Flp-H305L-3HA and test for loci immediately downstream of the DPC. Persistence of the DPC into S phase would lead to replication-borne one-ended DNA breaks. If you pull down a Flp-H305L-3HA molecule that has caused such a break, I would not expect to recover the downstream DNA sequences tested for in the qPCR. Did you normalize or control for this possibility in your quantification?
- Your SILAC-MS screen for Siz2-dependent sumoylation did not reveal any plausible DNA repair factor as SUMO target, and Top1 sumoylation was not affected by deletion of SIZ2. However, your MS data shows reduced sumoylation of several core histones (HHF1/HHF2, HTA1/HTA2, HTB1/HTB2) in the absence of Siz2. Would you expect extensive histone sumoylation to be in line with your proposed model of recruitment vs. exclusion of repair factors?
- Do you have evidence that the SUMO-dependent pathway choice could be conserved in mammals?
References:
Michel AH, Hatakeyama R, Kimmig P, Arter M, Peter M, Matos J, De Virgilio C & Kornmann BT (2017) Functional mapping of yeast genomes by saturated transposition. Elife 6: 1–28
Serbyn N, Noireterre A, Bagdiul I, Plank M, Michel AH, Loewith R, Kornmann B & Stutz F (2020) The Aspartic Protease Ddi1 Contributes to DNA-Protein Crosslink Repair in Yeast. Mol. Cell 77: 1066-1079.e9
doi: https://doi.org/10.1242/prelights.23401
Read preprintSign up to customise the site to your preferences and to receive alerts
Register hereAlso in the cell biology category:
Restoring mechanophenotype reverts malignant properties of ECM-enriched vocal fold cancer
Teodora Piskova
Germplasm stability in zebrafish requires maternal Tdrd6a and Tdrd6c
Justin Gutkowski
Leukocytes use endothelial membrane tunnels to extravasate the vasculature
Felipe Del Valle Batalla
Also in the genetics category:
Intracellular diffusion in the cytoplasm increases with cell size in fission yeast
Leeba Ann Chacko, Sameer Thukral
HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos
Anchel De Jaime Soguero
Significantly reduced, but balanced, rates of mitochondrial fission and fusion are sufficient to maintain the integrity of yeast mitochondrial DNA
Leeba Ann Chacko
Also in the molecular biology category:
Germplasm stability in zebrafish requires maternal Tdrd6a and Tdrd6c
Justin Gutkowski
Platelet-derived LPA16:0 inhibits adult neurogenesis and stress resilience in anxiety disorder
Harvey Roweth
Green synthesized silver nanoparticles from Moringa: Potential for preventative treatment of SARS-CoV-2 contaminated water
Safieh Shah, Benjamin Dominik Maier
preListscell biology category:
in theNovember in preprints – the CellBio edition
This is the first community-driven preList! A group of preLighters, with expertise in different areas of cell biology, have worked together to create this preprint reading lists for researchers with an interest in cell biology. Categories include: 1) cancer cell biology 2) cell cycle and division 3) cell migration and cytoskeleton 4) cell organelles and organisation 5) cell signalling and mechanosensing 6) genetics/gene expression
List by | Felipe Del Valle Batalla et al. |
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
List by | Alex Eve, Katherine Brown |
preLights peer support – preprints of interest
This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.
List by | preLights peer support |
The Society for Developmental Biology 82nd Annual Meeting
This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.
List by | Joyce Yu, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
List by | Alex Eve |
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
List by | Helen Zenner |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
List by | Nadja Hümpfer et al. |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
List by | Osvaldo Contreras |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
List by | Ana Dorrego-Rivas |
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
List by | Ana Dorrego-Rivas |
BioMalPar XVI: Biology and Pathology of the Malaria Parasite
[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria
List by | Dey Lab, Samantha Seah |
1
Cell Polarity
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
List by | Yamini Ravichandran |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
List by | Maiko Kitaoka et al. |
3D Gastruloids
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
List by | Paul Gerald L. Sanchez and Stefano Vianello |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
List by | Dey Lab |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
List by | Sandra Malmgren Hill |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
List by | Rob Hynds |
Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
List by | Pablo Ranea Robles |
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
List by | Dey Lab |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
List by | Sandra Franco Iborra |
Biophysical Society Annual Meeting 2019
Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA
List by | Joseph Jose Thottacherry |
ASCB/EMBO Annual Meeting 2018
This list relates to preprints that were discussed at the recent ASCB conference.
List by | Dey Lab, Amanda Haage |
Also in the genetics category:
End-of-year preprints – the genetics & genomics edition
In this community-driven preList, a group of preLighters, with expertise in different areas of genetics and genomics have worked together to create this preprint reading list. Categories include: 1) genomics 2) bioinformatics 3) gene regulation 4) epigenetics
List by | Chee Kiang Ewe et al. |
BSDB/GenSoc Spring Meeting 2024
A list of preprints highlighted at the British Society for Developmental Biology and Genetics Society joint Spring meeting 2024 at Warwick, UK.
List by | Joyce Yu, Katherine Brown |
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
List by | Reinier Prosee |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
List by | Sergio Menchero et al. |
Semmelweis Symposium 2022: 40th anniversary of international medical education at Semmelweis University
This preList contains preprints discussed during the 'Semmelweis Symposium 2022' (7-9 November), organised around the 40th anniversary of international medical education at Semmelweis University covering a wide range of topics.
List by | Nándor Lipták |
20th “Genetics Workshops in Hungary”, Szeged (25th, September)
In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf
List by | Nándor Lipták |
2nd Conference of the Visegrád Group Society for Developmental Biology
Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)
List by | Nándor Lipták |
EMBL Conference: From functional genomics to systems biology
Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020
List by | Jesus Victorino |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
List by | Maiko Kitaoka et al. |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
List by | Hiral Shah |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
List by | Sandra Malmgren Hill |
Zebrafish immunology
A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.
List by | Shikha Nayar |
Also in the molecular biology category:
2024 Hypothalamus GRC
This 2024 Hypothalamus GRC (Gordon Research Conference) preList offers an overview of cutting-edge research focused on the hypothalamus, a critical brain region involved in regulating homeostasis, behavior, and neuroendocrine functions. The studies included cover a range of topics, including neural circuits, molecular mechanisms, and the role of the hypothalamus in health and disease. This collection highlights some of the latest advances in understanding hypothalamic function, with potential implications for treating disorders such as obesity, stress, and metabolic diseases.
List by | Nathalie Krauth |
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
List by | Alex Eve, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
List by | Alex Eve |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
List by | Nadja Hümpfer et al. |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
List by | Ana Dorrego-Rivas |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar et al. |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
List by | Rob Hynds |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
List by | Sandra Franco Iborra |