Genetic analysis of RPA single-stranded DNA binding protein in Haloferax volcanii
Replication protein A (RPA) is a single-stranded DNA-binding protein that is present in all three domains of life. The roles of RPA include stabilising and protecting single- stranded DNA from nuclease degradation during DNA replication and repair. To achieve this, RPA uses an oligosaccharide-bindin...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
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2012
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| Online Access: | https://eprints.nottingham.ac.uk/12623/ |
| _version_ | 1848791543302848512 |
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| author | Stroud, A. L. |
| author_facet | Stroud, A. L. |
| author_sort | Stroud, A. L. |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Replication protein A (RPA) is a single-stranded DNA-binding protein that is present in all three domains of life. The roles of RPA include stabilising and protecting single- stranded DNA from nuclease degradation during DNA replication and repair. To achieve this, RPA uses an oligosaccharide-binding fold (OB fold) to bind single- stranded DNA.
Haloferax volcanii encodes three RPAs – RPA1, RPA2 and RPA3, of which rpa1 and rpa3 are in operons with genes encoding associated proteins (APs). The APs belong to the COG3390 group of proteins found in Euryarchaeota and feature an OB fold. Genetic analysis of deletion mutants was employed to determine if all three RPAs are essential for cell viability, and if there is an element of redundancy between RPA1 and RPA3. The hypothesis that the RPAs form a complex with their respective APs, as opposed to a heterotrimeric RPA complex, was also investigated. Furthermore, it was tested whether the RPAs and their respective APs are specific for each other, or whether they are interchangeable.
The genetic analysis showed that RPA2 is essential for cell viability, but that neither RPA1 nor RPA3 are. The rpa3, rpa3ap and the rpa3 operon deletion mutants showed sensitivity to DNA damage but only a slight growth defect. By contrast, the rpa1, rpa1ap, rpe and rpa1 operon mutants did not show any DNA damage sensitivity and an even milder growth defect. The double rpa1 rpa3 operon deletion was difficult to generate but unexpectedly lacked a significant DNA damage sensitivity and growth defect. The inability to make the double rpa1 rpa3ap and rpa1ap rpa3 deletion mutants suggests that the APs are specific for their respective RPAs.
Biochemical analysis involving histidine-tagged RPAs and APs was used to confirm the conclusions of the genetic analysis. The RPAs did not interact with each other, but instead co-purified with their respective APs. This finding reiterates that the RPAs do not form a heterotrimeric complex, as seen in eukaryotes, but instead form a novel complex with their respective APs. |
| first_indexed | 2025-11-14T18:30:11Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-12623 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T18:30:11Z |
| publishDate | 2012 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-126232025-02-28T11:20:22Z https://eprints.nottingham.ac.uk/12623/ Genetic analysis of RPA single-stranded DNA binding protein in Haloferax volcanii Stroud, A. L. Replication protein A (RPA) is a single-stranded DNA-binding protein that is present in all three domains of life. The roles of RPA include stabilising and protecting single- stranded DNA from nuclease degradation during DNA replication and repair. To achieve this, RPA uses an oligosaccharide-binding fold (OB fold) to bind single- stranded DNA. Haloferax volcanii encodes three RPAs – RPA1, RPA2 and RPA3, of which rpa1 and rpa3 are in operons with genes encoding associated proteins (APs). The APs belong to the COG3390 group of proteins found in Euryarchaeota and feature an OB fold. Genetic analysis of deletion mutants was employed to determine if all three RPAs are essential for cell viability, and if there is an element of redundancy between RPA1 and RPA3. The hypothesis that the RPAs form a complex with their respective APs, as opposed to a heterotrimeric RPA complex, was also investigated. Furthermore, it was tested whether the RPAs and their respective APs are specific for each other, or whether they are interchangeable. The genetic analysis showed that RPA2 is essential for cell viability, but that neither RPA1 nor RPA3 are. The rpa3, rpa3ap and the rpa3 operon deletion mutants showed sensitivity to DNA damage but only a slight growth defect. By contrast, the rpa1, rpa1ap, rpe and rpa1 operon mutants did not show any DNA damage sensitivity and an even milder growth defect. The double rpa1 rpa3 operon deletion was difficult to generate but unexpectedly lacked a significant DNA damage sensitivity and growth defect. The inability to make the double rpa1 rpa3ap and rpa1ap rpa3 deletion mutants suggests that the APs are specific for their respective RPAs. Biochemical analysis involving histidine-tagged RPAs and APs was used to confirm the conclusions of the genetic analysis. The RPAs did not interact with each other, but instead co-purified with their respective APs. This finding reiterates that the RPAs do not form a heterotrimeric complex, as seen in eukaryotes, but instead form a novel complex with their respective APs. 2012-07-17 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en arr https://eprints.nottingham.ac.uk/12623/1/Amy_Stroud_Final_corrected_version_.pdf Stroud, A. L. (2012) Genetic analysis of RPA single-stranded DNA binding protein in Haloferax volcanii. PhD thesis, University of Nottingham. |
| spellingShingle | Stroud, A. L. Genetic analysis of RPA single-stranded DNA binding protein in Haloferax volcanii |
| title | Genetic analysis of RPA single-stranded DNA binding protein in Haloferax volcanii |
| title_full | Genetic analysis of RPA single-stranded DNA binding protein in Haloferax volcanii |
| title_fullStr | Genetic analysis of RPA single-stranded DNA binding protein in Haloferax volcanii |
| title_full_unstemmed | Genetic analysis of RPA single-stranded DNA binding protein in Haloferax volcanii |
| title_short | Genetic analysis of RPA single-stranded DNA binding protein in Haloferax volcanii |
| title_sort | genetic analysis of rpa single-stranded dna binding protein in haloferax volcanii |
| url | https://eprints.nottingham.ac.uk/12623/ |