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Genetic and Biochemical Identification of a Novel Single-Stranded DNA-Binding Complex in Haloferax volcanii.

Stroud A, Liddell S, Allers T - Front Microbiol (2012)

Bottom Line: This indicates that the RPAs interact only with their respective associated proteins; this was corroborated by the inability to construct rpa1 rpap3 and rpa3 rpap1 double mutants.This is the first report investigating the individual function of the archaeal COG3390 RPA-associated proteins (RPAPs).We have shown genetically and biochemically that the RPAPs interact with their respective RPAs, and have uncovered a novel single-stranded DNA-binding complex that is unique to Euryarchaeota.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Queen's Medical Centre, University of Nottingham Nottingham, UK.

ABSTRACT
Single-stranded DNA (ssDNA)-binding proteins play an essential role in DNA replication and repair. They use oligonucleotide/oligosaccharide-binding (OB)-folds, a five-stranded β-sheet coiled into a closed barrel, to bind to ssDNA thereby protecting and stabilizing the DNA. In eukaryotes the ssDNA-binding protein (SSB) is known as replication protein A (RPA) and consists of three distinct subunits that function as a heterotrimer. The bacterial homolog is termed SSB and functions as a homotetramer. In the archaeon Haloferax volcanii there are three genes encoding homologs of RPA. Two of the rpa genes (rpa1 and rpa3) exist in operons with a novel gene specific to Euryarchaeota; this gene encodes a protein that we have termed RPA-associated protein (rpap). The rpap genes encode proteins belonging to COG3390 group and feature OB-folds, suggesting that they might cooperate with RPA in binding to ssDNA. Our genetic analysis showed that rpa1 and rpa3 deletion mutants have differing phenotypes; only Δrpa3 strains are hypersensitive to DNA damaging agents. Deletion of the rpa3-associated gene rpap3 led to similar levels of DNA damage sensitivity, as did deletion of the rpa3 operon, suggesting that RPA3 and RPAP3 function in the same pathway. Protein pull-downs involving recombinant hexahistidine-tagged RPAs showed that RPA3 co-purifies with RPAP3, and RPA1 co-purifies with RPAP1. This indicates that the RPAs interact only with their respective associated proteins; this was corroborated by the inability to construct rpa1 rpap3 and rpa3 rpap1 double mutants. This is the first report investigating the individual function of the archaeal COG3390 RPA-associated proteins (RPAPs). We have shown genetically and biochemically that the RPAPs interact with their respective RPAs, and have uncovered a novel single-stranded DNA-binding complex that is unique to Euryarchaeota.

No MeSH data available.


Related in: MedlinePlus

(A) Wild-type (WT), rpa1, rpap1, rpa1 operon, rpa3, rpap3, rpa3 operon (H195, H1217, H1216, H1246, H1244, H1410, and H1260, respectively), were plated out and exposed to ultraviolet (UV) radiation. The average and standard error (SE) of three experiments are shown. (B) The strains shown in (A) were plated out and exposed to mitomycin C (MMC). The average and SE of three experiments are shown.
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Figure 4: (A) Wild-type (WT), rpa1, rpap1, rpa1 operon, rpa3, rpap3, rpa3 operon (H195, H1217, H1216, H1246, H1244, H1410, and H1260, respectively), were plated out and exposed to ultraviolet (UV) radiation. The average and standard error (SE) of three experiments are shown. (B) The strains shown in (A) were plated out and exposed to mitomycin C (MMC). The average and SE of three experiments are shown.

Mentions: Both eukaryotic and bacterial SSB are involved in DNA repair. To examine if H. volcanii RPA1 and RPA3 function in DNA repair, the effects of DNA damage on cell survival of H1217 and H1244 were examined. UV irradiation results in the formation of cyclobutane pyrimidine dimers and 6-4 pyrimidine-pyrimidone dimer photoproducts, as well as ssDNA nicks that indirectly generate double-stranded DNA breaks (DSBs). The latter require repair by homologous recombination (HR) or single-strand DNA annealing (Fousteri and Mullenders, 2008; Rouillon and White, 2011). MMC is a chemotherapeutic agent that reacts with DNA generating covalent interstrand cross-links, requiring removal by nucleotide excision repair (NER) and HR (Tomasz et al., 1987). The Δrpa1 mutant H1217 was no more sensitive than the WT to UV and MMC-induced DNA damage, however the Δrpa3 mutant H1244 exhibited moderate sensitivity to both UV and MMC-induced DNA damage (Figure 4).


Genetic and Biochemical Identification of a Novel Single-Stranded DNA-Binding Complex in Haloferax volcanii.

Stroud A, Liddell S, Allers T - Front Microbiol (2012)

(A) Wild-type (WT), rpa1, rpap1, rpa1 operon, rpa3, rpap3, rpa3 operon (H195, H1217, H1216, H1246, H1244, H1410, and H1260, respectively), were plated out and exposed to ultraviolet (UV) radiation. The average and standard error (SE) of three experiments are shown. (B) The strains shown in (A) were plated out and exposed to mitomycin C (MMC). The average and SE of three experiments are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376784&req=5

Figure 4: (A) Wild-type (WT), rpa1, rpap1, rpa1 operon, rpa3, rpap3, rpa3 operon (H195, H1217, H1216, H1246, H1244, H1410, and H1260, respectively), were plated out and exposed to ultraviolet (UV) radiation. The average and standard error (SE) of three experiments are shown. (B) The strains shown in (A) were plated out and exposed to mitomycin C (MMC). The average and SE of three experiments are shown.
Mentions: Both eukaryotic and bacterial SSB are involved in DNA repair. To examine if H. volcanii RPA1 and RPA3 function in DNA repair, the effects of DNA damage on cell survival of H1217 and H1244 were examined. UV irradiation results in the formation of cyclobutane pyrimidine dimers and 6-4 pyrimidine-pyrimidone dimer photoproducts, as well as ssDNA nicks that indirectly generate double-stranded DNA breaks (DSBs). The latter require repair by homologous recombination (HR) or single-strand DNA annealing (Fousteri and Mullenders, 2008; Rouillon and White, 2011). MMC is a chemotherapeutic agent that reacts with DNA generating covalent interstrand cross-links, requiring removal by nucleotide excision repair (NER) and HR (Tomasz et al., 1987). The Δrpa1 mutant H1217 was no more sensitive than the WT to UV and MMC-induced DNA damage, however the Δrpa3 mutant H1244 exhibited moderate sensitivity to both UV and MMC-induced DNA damage (Figure 4).

Bottom Line: This indicates that the RPAs interact only with their respective associated proteins; this was corroborated by the inability to construct rpa1 rpap3 and rpa3 rpap1 double mutants.This is the first report investigating the individual function of the archaeal COG3390 RPA-associated proteins (RPAPs).We have shown genetically and biochemically that the RPAPs interact with their respective RPAs, and have uncovered a novel single-stranded DNA-binding complex that is unique to Euryarchaeota.

View Article: PubMed Central - PubMed

Affiliation: School of Biology, Queen's Medical Centre, University of Nottingham Nottingham, UK.

ABSTRACT
Single-stranded DNA (ssDNA)-binding proteins play an essential role in DNA replication and repair. They use oligonucleotide/oligosaccharide-binding (OB)-folds, a five-stranded β-sheet coiled into a closed barrel, to bind to ssDNA thereby protecting and stabilizing the DNA. In eukaryotes the ssDNA-binding protein (SSB) is known as replication protein A (RPA) and consists of three distinct subunits that function as a heterotrimer. The bacterial homolog is termed SSB and functions as a homotetramer. In the archaeon Haloferax volcanii there are three genes encoding homologs of RPA. Two of the rpa genes (rpa1 and rpa3) exist in operons with a novel gene specific to Euryarchaeota; this gene encodes a protein that we have termed RPA-associated protein (rpap). The rpap genes encode proteins belonging to COG3390 group and feature OB-folds, suggesting that they might cooperate with RPA in binding to ssDNA. Our genetic analysis showed that rpa1 and rpa3 deletion mutants have differing phenotypes; only Δrpa3 strains are hypersensitive to DNA damaging agents. Deletion of the rpa3-associated gene rpap3 led to similar levels of DNA damage sensitivity, as did deletion of the rpa3 operon, suggesting that RPA3 and RPAP3 function in the same pathway. Protein pull-downs involving recombinant hexahistidine-tagged RPAs showed that RPA3 co-purifies with RPAP3, and RPA1 co-purifies with RPAP1. This indicates that the RPAs interact only with their respective associated proteins; this was corroborated by the inability to construct rpa1 rpap3 and rpa3 rpap1 double mutants. This is the first report investigating the individual function of the archaeal COG3390 RPA-associated proteins (RPAPs). We have shown genetically and biochemically that the RPAPs interact with their respective RPAs, and have uncovered a novel single-stranded DNA-binding complex that is unique to Euryarchaeota.

No MeSH data available.


Related in: MedlinePlus