DNAPRO - Deciphering DNA-Protein Crosslink Repair in vivo using zebrafish model

DNAPRO - Deciphering DNA-Protein Crosslink Repair in vivo using zebrafish model

SPRTN protease core model

Datum početka projekta
Datum kraja projekta
Kategorija projekta
Projekti Hrvatske zaklade za znanost

Project summary

DNA-protein crosslink (DPC) is a type of DNA lesion where a protein becomes irreversibly covalently bound to DNA upon exposure to endogenous or exogenous crosslink inducers. Endogenous DPC inducers are products of normal cellular metabolism such as reactive oxygen species, aldehydes and DNA helical alterations, while exogenous inducers include UV light, ionizing radiation and various chemicals. DNA-protein crosslinks are common DNA lesions which present a physical blockage to all DNA transactions: replication, transcription, recombination and repair. If not repaired, DPCs cause genomic instability and adverse phenotypes in humans including premature aging, neurodegeneration and cancer. Despite the frequency and severe outcomes of DPCs, DNA-protein crosslink repair (DPCR) has been sparsely studied, mostly because it has not been considered a separate DNA damage repair pathway until recently. In 2014 and 2016, several groups have identified a novel proteases, Wss1 and SPRTN, which initiate the removal of DPCs through the proteolytic digestion of crosslinked proteins. The discovery of proteolysis-coupled DPC repair lead to recognition of the DNA-protein crosslink repair as a separate DNA damage repair pathway. However, we currently do not know how is the pathway orchestrated and which other factors are involved, while almost nothing is known of DPCR mechanism in vivo. Therefore, within this project we aim to unravel the orchestration of the DPCR pathway in vivousing zebrafish (Danio rerio) as a well-characterized vertebrate model. We will use CRISPR/Cas9 gene manipulation tools to knock-out or mutate specific genes in zebrafish which we suspect are involved in the removal of DNA-protein crosslinks. Contribution of each protein (and their combinations) to the DNA-protein crosslink repair will be quantified after DPC isolation from transgenic zebrafish embryos and adults. We will also generate a GFP reporter assay in transgenic fish which will enable the quantification of DPCR efficiency in vivo.

DNA damage and repair

Loss of DNA integrity causes cancer, neurodegeneration and aging. The development, growth and maintenance of all tissues in an organism relies on accurate and error-free DNA replication and transcription. Therefore, elaborate and complex mechanisms are in place to cope with DNA damage. DNA is constantly exposed to various endogenous (products of cellular metabolism) and exogenous (environmental) sources of damage including: oxidative radicals, aldehydes, UV light, ionizing radiation (IR) and pharmaceuticals. Each of the mentioned damage sources create a specific set of DNA lesions including single and double strand DNA breaks (SSB and DSBs), apurinic/apyrimidinic (AP) sites, intra and interstrand DNA-DNA crosslinks (ICLs) and DNA-protein crosslinks(DPCs). If cells cannot repair the damaged DNA, or if this repair is erroneous, the consequences are severe and include apoptosis, senescence and/or genomic instability which in turn can lead to cancer, accelerated aging and neurodegeneration. Over the last 30 years, intense research efforts have been directed towards understanding the DNA repair mechanisms that cope with the diversity of DNA lesions. Currently, we know that SSBs are repaired by base excision repair (BER) and nucleotide excision repair (NER), DSBs are repaired by non-homologous end joining (NHEJ), homologous recombination (HR) and alternative NHEJ (alt-NHEJ), and ICLs are repaired by the Fanconi Anemia pathway. However, how DPC repair is orchestrated, is currently unknown. In 2015, the pioneers of DNA repair research were awarded the Nobel Prize for Chemistry, testifying to the importance of understanding cellular DNA repair pathways.

DNA-protein crosslinks

DNA-protein crosslinks (DPCs) are a common type of DNA damage which appear when a protein forms an irreversible covalent bond with the DNA. Under physiological conditions these lesions are caused by reactive oxygen and nitrogen species, DNA helical alterations and increased aldehyde concentration due to histone demethylation, AlkB-type repair, amino acid metabolism, and lipid peroxidation. DPCs are also induced by exogenous sources such as UV light, ionizing radiation and chemotherapeutics. These lesions are very diverse because any protein in proximity to DNA can be crosslinked upon exposure to endogenous or exogenous crosslinking sources. DPCs are also intricately complex due to the diversity of crosslinking chemistries and protein sizes.

DPCs present aphysical blockage toall DNA transactions: replication, transcription, recombination and repair and therefore the consequences of impairedDNA-Protein Crosslink Repair (DPCR)are severe. Considering their frequent occurrence and detrimental effect on all DNA transactions, it is not surprising that DPCs are implicated in aging, cardiovascular diseases, neurodegeneration and cancer. On a cellular level, aberrant DPC repair leads to the formation of DSBs, genomic instability and/or cell death, while on the organismal level impaired DPCR was so far shown to cause premature aging phenotypes and cancer.

Proteolysis-dependent DNA-Protein Crosslink Repair

The mechanism of DNA-Protein Crosslink Repair (DPCR) is still largely unknown. The reason is in part due to the misconception in the field that DPCs are repaired by canonical DNA damage repair pathways, nucleotide excision repair (NER) and homologous recombination (HR). However, we and others have recently shown that DPC repair is a specialised DNA damage repair pathway which relies on the proteolytic digestion of crosslinked proteins. The central players in the pathway are the metalloproteases Wss1 in yeast andSPRTN(or DVC1) in mammals which initiate DPCR by the proteolytic cleavage of crosslinked protein, followed by the removal of the protein remnant from DNA backbone via different downstream factors. However, the discovery of the SPRTN protease only scratches the surface of this pathway and the vast majority of questions including the identity of other DPCR factors still remain to be answered. Indeed, all known DNA repair pathways involve a complex machinery consisting of numerous proteins. For example, both homologous recombination-mediated DSB repair and Nucleotide excision repair (NER) each involve >30 proteins.

Other factors with a role in DNA-Protein Crosslink Repair

Emerging evidence suggest that three key factors, tyrosyl phosphodiesterases (TDPs), Nucelotide excision repair (NER) and nuclease Mre11 have far broader roles in DPCR than initially assumed.

Specific, enzymatic type of DPCs, Topoisomerase 1 and 2 cleavage complexes (TOP1ccs and TOP2ccs) are in part repaired by tyrosyl phosphodiesterases1 (TDP1) and 2 (TDP2). These enzymes require upstream proteolysis of DPCs, which reduces the size of crosslinked topoisomerases. TDPs then remove the protein remnant from the DNA backbone by incising the DNA around the DPC remnant with their esterase activity. However,in vitro studies [36] suggest that TDPs might not only remove TOPccs, but also other DPC types. Research into these areas has been hindered by the lack of adequate models and we aim to answer the open question with an in vivo model using zebrafish transgenic strains.

Small DPCs(below 8-16 kDa) can be repaired by Nucleotide excision repair pathway (NER). However, direct removal of DPCs via NER machinery has only been shown in vitro and in cell lines. In vivo studies on the organismal level which will ultimately prove under which conditions NER is involved in DPC repair are currently lacking.

The role of homologous recombination (HR) in the DPC repair is still unclear because it is difficult to distinguish the role of HR in DPC removal from the canonical role of HR in DSB repair given that unrepaired DPCs eventually cause DSBs. Recently it was shown that a nucleaseMRE11, known primarily as a component of the MRN complex, a central part of the HR pathway, can remove TOP2ccs and artificially crosslinked streptavidin protein via the endonucleolytic cleavage of DNA near the protein crosslink. It is still unclear whether this action of MRE11 is HR-dependent  or HR-independent. In any case, MRE11 is a novel player in DPC repair and we will investigate its connection to SPRTN-mediated DPC proteolysis and its overall significance and specificity in the DPC removal in vivo.

Unknown factors in DPC repair

Considering that SPRTN is a replication-specific protease, it is probable that another protease acts in lowly proliferative cells where DPCs pose a threat to transcription progression. Indeed, it was shown that the removal of TOP1ccs via TDP1 happens in non-proliferative cells. Considering that proteolysis is a prerequisite for TDP1-mediated peptide remnant removal, we searched the genome for other SPRTN-like protease. Our phylogenetic analysis of the SPRT family in metazoans indeed identified a SPRT-like protein family,ACRC(Popovic, unpublished data). ACRC could be the protease operating in non-replicative cells, and this question will be addressed within the proposed project.

p97is an AAA ATPase (ATPase associated with various activities) involved in many cellular processes including ERAD (Endoplasmatic Reticulum Associated Degradation), vesicular trafficking, DNA replication and DNA repair. Considering that p97 can extract proteins from chromatin through its ATP-dependent segregase activity and that SPRTN forms a stable complex with p97 and was in fact initially identified as a p97 cofactor, we hypothesize that p97 could act downstream of SPRTN proteolysis by extracting protein remnants from the DNA backbone.

Zebrafish as a research model

Zebrafish is a well-established vertebrate model in developmental biology, toxicology and biomedicine. It offers a uniquely elegant toolbox to investigate the orchestration of DPC repair in vivo due to the ease of genetic manipulation and very fast developmental cycle. Additional advantages of the model include easy and automated fish maintenance, established methods for forward and reverse genetic studies and high quality of sequenced genome. Indeed, zebrafish is the only vertebrate whose genome is sequenced with similar quality to those of human and mouse. Additionally, given the high degree of evolutionary conservation between zebrafish and mammals, especially in respect to DNA damage related genes, utilizing the zebrafish model to understand the mechanisms of DNA repair pathwaysis an emerging field with proven benefits.

Besides being an excellent model to decipher DPCR orchestration n vivo, we will use zebrafish to develop a toolbox which will enable the quantification of SPRTN-mediated initiation of DPCR in vivo.Currently, such a system does not exist, because researchers were focused on developing approaches to measure DPCs, rather than DPCR repair. The developed toolbox will enable us to better understand the pathway on an organismal level, and to assess the effects of chemical agents on DPC repair capacity.

The overall aim of this project is to investigate the mechanisms of DPC repair on the organismal level. Specifically,

  1. Factors acting downstream of SPRTN by removal of protein remnant:

     a)     Analyzing the involvement of NER, TDP1, TDP2 and p97 using zebrafish models

  1. NER deficient zebrafish: XPA knock-out
  2. TDP1 or TDP2 catalytic dead mutant zebrafish
  3. P97 deficient zebrafish: mutant alleles of p97
  4. Other factors actingwith SPRTN:

     b)     Analyzing the role of ACRC and MRE11 using zebrafish model

  1. ACRC catalytic dead zebrafish
  2. MRE11 nuclease-deficient fish: conditional mutant alleles of Mre11
Ostali suradnici

dr.sc. Ivan Mihaljevic - postdoctoral researcher

MSc Christine Supina (mag.mol.bio.) - PhD student


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