Biotechnology with a pinch of salt - isolation of DNA repair enzymes from halophilic archaea
Lead Research Organisation:
University of Nottingham
Department Name: School of Life Sciences
Abstract
Background
Hel308 is a monomeric 3' to 5' DNA helicase conserved throughout archaea and metazoans however, is absent from bacteria and fungi. Hel308 is a member of the helicase superfamily 2. An investigation into Drosophila melanogaster mutants which exhibited hypersensitivity to DNA crosslinking agents led to the discovery of Hel308 and consequently the human homologue HELQ. Dependant on the organism of study, Hel308 is known by other names including HELQ in higher eukaryotes, and Hjm in some archaeal species for example Purococcus furiosus and Sulfolobus tokodaii. Though a member of the Ski2 family of helicases, Hel308 acts analogously to RecQ DNA family helicases and has consequently been implicated in a variety of overlapping DNA repair pathways, as well as homologous recombination, and genome stability. Despite these connections, the exact role of Hel308 has remained elusive.
Outline
Structural, biochemical, and genetic analyses of Hel308 and its respective gene, have started to unravel the Hel308 mystery. Utilizing both the biochemical and genetic tool-boxes of Haloferax volcanii, protein and genetic interacting partners of Hel308 will be further explored under both native and stress conditions. Additionally, mutations of key residues identified via structural and sequence analysis will be investigated for phenotypic consequences. Overall, the function(s) of and the regulatory mechanism(s) of Hel308 will be examined.
Unfortunately, no crystal structure for the Haloferax volcanii Hel308 has been obtained to date, there are however resolved crystal structures of three Hel308 homologues. From these crystal structures Hel308 is shown to be composed of 5 domains around a central DNA binding core which is identifiable by a lining of essential DNA binding residues. The 3' ssDNA overhang threads through this DNA binding core and the five domains drive a 13-hairpin between the two strands subsequently separating the DNA duplex. As with all superfamily 2 helicases Hel308 requires ATP binding and hydrolysis to enable translocation along the DNA. Key residues targeted for mutation include Walker A and B ATPase dead mutations, both independently and in combination with the previously identified hyper-recombinant point mutations.
It has recently been shown that in Haloferax volcanii, mutants deleted for radB exhibit a decrease in the level of homologous recombination to approximately 5% of that in wild type cells. However, a double deletion of hel308 with radB restores, in part, homologous recombination. This data suggests an anti-recombinase role for Hel308. Further genetic analysis between hel308 and the homologous recombination pathway will be conducted focussing on the relationship between RadA and Hel308.
Impact
The mammalian Hel308 homologue HELQ, has previously been implicated in genome stability upon the recognition of several certain Hel308 single nucleotide polymorphisms (SNPs) being more prevalent in numerous types of head and neck cancers including esophageal squamous cell carcinoma and gastric adenocarcinoma. In addition to this, it has been suggested that HELQ has a critical role in germ cell maintenance and tumour suppression of ovarian cancers in mammals, indicating a possible role in replication-coupled DNA repair.
Due to the evolutionary descent of eukaryotes from archaea, archaea present an opportunity to explore the function of eukaryotic HELQ in a simplified system. By exploring the function of Hel308 in Haloferax volcanii, we can begin to elucidate the mechanism of HELQ and therefore understand better how HELQ affects genome stability and DNA repair. Ultimately this understanding could lead to improved treatments for these cancers.
Hel308 is a monomeric 3' to 5' DNA helicase conserved throughout archaea and metazoans however, is absent from bacteria and fungi. Hel308 is a member of the helicase superfamily 2. An investigation into Drosophila melanogaster mutants which exhibited hypersensitivity to DNA crosslinking agents led to the discovery of Hel308 and consequently the human homologue HELQ. Dependant on the organism of study, Hel308 is known by other names including HELQ in higher eukaryotes, and Hjm in some archaeal species for example Purococcus furiosus and Sulfolobus tokodaii. Though a member of the Ski2 family of helicases, Hel308 acts analogously to RecQ DNA family helicases and has consequently been implicated in a variety of overlapping DNA repair pathways, as well as homologous recombination, and genome stability. Despite these connections, the exact role of Hel308 has remained elusive.
Outline
Structural, biochemical, and genetic analyses of Hel308 and its respective gene, have started to unravel the Hel308 mystery. Utilizing both the biochemical and genetic tool-boxes of Haloferax volcanii, protein and genetic interacting partners of Hel308 will be further explored under both native and stress conditions. Additionally, mutations of key residues identified via structural and sequence analysis will be investigated for phenotypic consequences. Overall, the function(s) of and the regulatory mechanism(s) of Hel308 will be examined.
Unfortunately, no crystal structure for the Haloferax volcanii Hel308 has been obtained to date, there are however resolved crystal structures of three Hel308 homologues. From these crystal structures Hel308 is shown to be composed of 5 domains around a central DNA binding core which is identifiable by a lining of essential DNA binding residues. The 3' ssDNA overhang threads through this DNA binding core and the five domains drive a 13-hairpin between the two strands subsequently separating the DNA duplex. As with all superfamily 2 helicases Hel308 requires ATP binding and hydrolysis to enable translocation along the DNA. Key residues targeted for mutation include Walker A and B ATPase dead mutations, both independently and in combination with the previously identified hyper-recombinant point mutations.
It has recently been shown that in Haloferax volcanii, mutants deleted for radB exhibit a decrease in the level of homologous recombination to approximately 5% of that in wild type cells. However, a double deletion of hel308 with radB restores, in part, homologous recombination. This data suggests an anti-recombinase role for Hel308. Further genetic analysis between hel308 and the homologous recombination pathway will be conducted focussing on the relationship between RadA and Hel308.
Impact
The mammalian Hel308 homologue HELQ, has previously been implicated in genome stability upon the recognition of several certain Hel308 single nucleotide polymorphisms (SNPs) being more prevalent in numerous types of head and neck cancers including esophageal squamous cell carcinoma and gastric adenocarcinoma. In addition to this, it has been suggested that HELQ has a critical role in germ cell maintenance and tumour suppression of ovarian cancers in mammals, indicating a possible role in replication-coupled DNA repair.
Due to the evolutionary descent of eukaryotes from archaea, archaea present an opportunity to explore the function of eukaryotic HELQ in a simplified system. By exploring the function of Hel308 in Haloferax volcanii, we can begin to elucidate the mechanism of HELQ and therefore understand better how HELQ affects genome stability and DNA repair. Ultimately this understanding could lead to improved treatments for these cancers.
Organisations
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M008770/1 | 01/10/2015 | 31/10/2024 | |||
| 1645070 | Studentship | BB/M008770/1 | 01/10/2015 | 30/09/2019 |
| Description | Hel308 is a monomeric 3' to 5' DNA helicase conserved throughout archaea and metazoans, but is absent from bacteria and fungi. Hel308 acts analogously to RecQ-family helicases, which serve as guardians of genome stability. Hel308 has been implicated in a variety of overlapping DNA repair pathways, including homologous recombination. Hel308 localises at damaged DNA replication forks in both humans and archaea. However, the exact role of Hel308 remains elusive. Haloferax volcanii was used as a model archaeon to study cellular role of Hel308. Deletion mutants of hel308, or point mutants that affect the ATP- dependent helicase activity, are slow-growing and are sensitive to DNA cross- linking agents, which are potent blocks to DNA replication. By contrast, point mutants that exhibit hyper-recombinant phenotypes are not deficient in DNA repair or normal growth. This study reiterated the involvement of Hel308 in homologous recombination (HR), mismatch repair (MMR), nucleotide excision repair (NER), and transcription coupled repair (TCR). Most interestingly, the overexpression of Hel308 (WT) and Hel308-D145N yields little of the target protein but co-purifies a large quantity of a 30kDa protein identified by mass spectrometry as DlnA (HVO_2382). DlnA is found in the highly conserved RadB neighbourhood and belongs to the YqgF family of endonucleases. In E. coli, YqgF has been proposed to function as an alternative to RuvC (Aravind, Makarova and Koonin, 2000), which acts to resolve Holliday junctions (Rocha, Cornet and Michel, 2005). In H. volcanii, the role of DlnA is yet to be determined but this interaction with Hel308 may give further insight to how both proteins function. For this reason, it would be of interest to repeat the overexpression of Hel308 in a strain deleted for DlnA. Deleting the hel308 gene increases recombination but decreases DNA repair and growth. This suggests that Hel308 actively targets and unwinds D-loops, supporting the proposal that Hel308 functions as an anti-recombinase. The point mutants F316A and R743A resulted in ~166,000-fold and ~35,500-fold increase in recombination frequency respectively, without affecting DNA repair or growth. This result is due exclusively to increased non-crossover recombination, since not a single crossover event was observed for these strains. The synthesis dependent strand annealing (SDSA) pathway produces non-crossover products, which reduces the likelihood of a genome rearrangement (San Filippo, Sung and Klein, 2008). Therefore, Hel308 regulates the levels of recombination and may also influence the pathway choice by which the recombination intermediates are resolved. It would be of interest to determine how Hel308 is involved in this pathway choice. Strains deleted for hel308 exhibit significant fitness defects and are sensitive to MMC treatment and sometimes UV irradiation, depending on the genotypic background. Mutating residues F316 and R743 increases recombination without affecting DNA repair or growth. By contrast, ATPase-null (hel308-D145N) strains exhibit a decrease in recombination levels to half that of wild type and exhibit ?hel308-like phenotype regarding DNA repair and growth. The hyper- recombinant point mutations identify a separation of function of Hel308, where presumably the ability to bind D-loop intermediates of recombination is affected, but the helicase activity involved in DNA repair is not. To further explore this separation of function, it would also be of interest to couple the hyper- recombinant and ATPase-null point mutations in Hel308 with the deletion of the recombination genes radA and radB, as well as the 5-gene radB neighbourhood deletion (?rcrA?radB?ndnR?hrp?cdc48a). Replacing hel308 with the ATPase-null allele hel308-D145N decreases recombination levels to half that of wild type. The combined ATPase-null and hyper-recombinant mutations, hel308-D145N-F316A and hel308-D145N- R743A, also decrease recombination levels to half that of wild type. The decrease in recombination when the D145N mutation is present implies that helicase activity is important for homologous recombination; conversely, the increase in recombination in the ?hel308 strain contradicts this. Walker A mutations (K53A/R) were not viable and possibly lethal to the cell, whereas the Walker B mutation (D145N) was viable and showed a ?hel308-like phenotype in terms of DNA repair and growth. This suggests that Hel308 first binds DNA tightly. On binding ATP, Hel308 changes conformation and loosens its grip on the DNA but remains static. Hydrolysis of the ATP causes a second conformational change and a release of energy, resulting in a paddling motion and translocation along the DNA. To further explore the role of Hel308 in homologous recombination, hel308 was successfully deleted in combination with the homologous recombination gene radA. Strains deleted for radA usually have a severe growth defect and are very sensitive to both UV irradiation and MMC treatment. Strains deleted for hel308 exhibit similar phenotypes, but not to the same severity as the ?radA strains. Most interestingly, the strain deleted for both hel308 and radA generated in this study was healthier than either single deletion. These data suggest that in the absence of Hel308-mediated repair and homologous recombination, a third distinct DNA-repair pathway is activated, which is ordinarily supressed by both Hel308-mediated repair and homologous recombination. An alternative explanation is that in deleting both the hel308 and radA genes, a suppressor mutation has arisen. Only a singular clone with the ?hel308?radA phenotype was generated, which supports the suppressor-mutation theory. However, a suppressor mutation or mutations would need the ability to bypass two distinct repair pathways in order to restore wildtype-like fitness, which seems unlikely. In order to determine whether a suppressor mutation or mutations have arisen during the deletion of hel308 and radA, analysis of the H3515 strain via whole genome sequencing needs to be completed. Complementation assays, reintroducing hel308 and radA at the respective chromosomal loci, would also aid in the understanding of the complex interplay between these pathways. Furthermore, it would be of interest to reattempt the triple deletion of hel308, radB, and radA. |
| Exploitation Route | In this study, the Walker A mutations (K53A/R) were not viable and possibly lethal to the cell, whereas the Walker B mutation (D145N) was viable and had a ?hel308-like phenotype in terms of DNA repair and growth. If ATP is bound prior to DNA binding, then the Walker A mutation should also give rise to a ?hel308-like phenotype in terms of DNA repair and growth. The K53A/R mutations are more detrimental to the cell than the deletion of the entire gene suggesting that Hel308 first binds DNA tightly. On binding ATP, Hel308 changes conformation and loosens its grip on the DNA but remains static. Hydrolyses of the ATP causes a second conformational change and a release of energy resulting in the paddling motion and translocation along the DNA. Unable to bind ATP nor translocate, the Hel308-K53A/R affectively creates a 'road-block' by binding tightly to DNA and stalling replication and repair. This results in catastrophic genome arrest, hence the cells are not viable. Whilst this study attempted to explore this, the correct strain for this episomal expression analysis was not generated. Therefore, it would be of interest to repeat the episomal expression assay with the correct strain in order to determine whether or not the Walker A mutant is lethal. During this study, several attempts to combine the hyper-recombinant and ATPase-null point mutations in Hel308 with either the deletion of radB alone or the 5-gene radB neighbourhood deletion (?rcrA?radB?ndnR?hrp?cdc48a) were made but remained unsuccessful. In H. volcanii, a low level of recombination, ~5% that of wildtype, still occurs within radB deleted strains. Therefore, introducing the Hel308 hyper-recombinant and ATPase-null point mutations into ?radB and ?rcrA?radB?ndnR?hrp?cdc48a genetic backgrounds, would indicate whether Hel308 is regulating homologous recombination via direct interaction with RadB or by other means. It would also be of interest to couple the hyper-recombinant and ATPase-null point mutations in Hel308 with the deletion of radA. This study has revealed interactions of Hel308 with proteins involved in DNA repair, replication, and RNA processing. To confirm these interactions, repetition of the protein:protein interaction assays alongside an empty vector control is required. Genuine interactions may also be determined by the reverse protein:protein interaction assays using tagged versions of the potential interaction partners. Identification of the presence of Hel308 via mass spectrometry would provide further evidence that the interaction in vivo is genuine. In order to determine whether a suppressor mutation or mutations have arisen during the deletion of hel308 and radA, analysis of the H3515 strain via Next Gen sequencing needs to be completed. Complementation assays, reintroducing hel308 and radA at the respective chromosomal loci, would also aid in the understanding of the complex interplay between these pathways. In order to achieve this, the radA in trans expression construct, pTA635 (or the radA-S101P in trans expression construct, pTA2080), must be introduced before the normal transformation protocol can be attempted. Where possible, it would be of interest to delete the genes encoding the interaction partners of Hel308 in combination with hel308. Inducible complementation assays should be used where the combination of deleted genes appears to be synthetically lethal. Growth, DNA damage, and recombination phenotypes of viable strains should also be determined. These analyses would give further insight and aid understanding of the role of Hel308 in DNA repair, replication, and RNA processing. |
| Sectors | Pharmaceuticals and Medical Biotechnology,Other |
| Description | A level student visit |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Schools |
| Results and Impact | Around fifteen year 12 (A level) students visited the Archaea Lab, I was responsible for giving them a tour and answering any questions they had about the research we did, what it was like to work in a lab etc. The aim was to inspire the next generation of scientists and widening the students awareness of scientific careers available. |
| Year(s) Of Engagement Activity | 2018 |
| Description | Mansfield Science Fair |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Schools |
| Results and Impact | Alongside two other students I ran stall at Mansfield Science Fair on "Crack the Code". The aim of the activity is to explain protein production, linking DNA, RNA and protein. To do this we will have a race with leader board getting the students to transcribe a short DNA sequence into mRNA and then translate it into amino acid codons. We will also show how we use DNA, mRNA and proteins in the lab on a regular basis. Students engaged very well, even when seemingly uninterested or overwhelmed by the subject material, they engaged in the competition and learned in the process. I enjoyed when the students were genuinely interested in the subject, but I also enjoyed the atmosphere of competition with different teams coming back and checking they were still on top and having another go with a different code if not. It was sometimes difficult to encourage the pupils into having a go, many thought it was a very complicated subject area and beyond their capabilities. It was also very apparent the differences in curriculum between the schools, though all year 10 some pupils obviously had learned a bit about DNA base pairing etc however for others this was a complete alien subject for them, for this reason pitching our activity to the passers-by was more difficult than anticipated as we were never sure what they did and didn't know - we originally devised a pitch based on the level the BBC bite size website suggested they would be at, and though accurate in some cases, it didn't hold true for all. As a whole I thought the activity was a success but perhaps I would invest some time into making it more accessible by either simplifying the problems or by introducing the activity in a more fun and relevant manner. I would also like to include 3D models of DNA helicase, the single strand mRNA and of a protein so that the students can truly appreciate what the cell is achieving every second. |
| Year(s) Of Engagement Activity | 2016 |