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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

H. volcanii strains containing (A) histidine-tagged RPA1 (H1482), (B) histidine-tagged RPAP1 (H1483), (C) histidine-tagged RPA3 (H1480), and (D) histidine-tagged RPAP3 (H1481) were used in protein overexpression. The histidine-tagged recombinant proteins were purified from the soluble fraction (lysate) by affinity chromatography on a Ni2+ chelating column, samples were taken from the flow-through (FT) and bound proteins were eluted using 50 and 500 mM imidazole. Precipitation using trichloroacetic acid and deoxycholate was used to enhance visualization and identification of the eluted proteins by mass spectrometry. Stars denote contaminants shown in Figure 5D.
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Figure 6: H. volcanii strains containing (A) histidine-tagged RPA1 (H1482), (B) histidine-tagged RPAP1 (H1483), (C) histidine-tagged RPA3 (H1480), and (D) histidine-tagged RPAP3 (H1481) were used in protein overexpression. The histidine-tagged recombinant proteins were purified from the soluble fraction (lysate) by affinity chromatography on a Ni2+ chelating column, samples were taken from the flow-through (FT) and bound proteins were eluted using 50 and 500 mM imidazole. Precipitation using trichloroacetic acid and deoxycholate was used to enhance visualization and identification of the eluted proteins by mass spectrometry. Stars denote contaminants shown in Figure 5D.

Mentions: Histidine-tagged RPA1 and RPA3 pulled down their respective RPAPs, and histidine-tagged RPAP1 and RPAP3 pulled down their respective RPAs (Figure 6). However, histidine-tagged RPA1 did not pull down RPAP3, and vice versa. This confirms that the RPAs interact specifically with their respective RPAPs, supporting our conclusions based on the failure to generate Δrpa1 Δrpap3 and Δrpa3 Δrpap1 deletion mutants. Neither RPA1 nor RPA3 pulled down RPA2, and histidine-tagged RPA2 did not pull down RPA1 or 3, or either of the RPAPs (data not shown). This supports the suggestion that the three RPAs of H. volcanii do not form a heterotrimer as observed in eukaryotes and P. furiosus, but instead form three separate ssDNA-binding factors.


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

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

H. volcanii strains containing (A) histidine-tagged RPA1 (H1482), (B) histidine-tagged RPAP1 (H1483), (C) histidine-tagged RPA3 (H1480), and (D) histidine-tagged RPAP3 (H1481) were used in protein overexpression. The histidine-tagged recombinant proteins were purified from the soluble fraction (lysate) by affinity chromatography on a Ni2+ chelating column, samples were taken from the flow-through (FT) and bound proteins were eluted using 50 and 500 mM imidazole. Precipitation using trichloroacetic acid and deoxycholate was used to enhance visualization and identification of the eluted proteins by mass spectrometry. Stars denote contaminants shown in Figure 5D.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: H. volcanii strains containing (A) histidine-tagged RPA1 (H1482), (B) histidine-tagged RPAP1 (H1483), (C) histidine-tagged RPA3 (H1480), and (D) histidine-tagged RPAP3 (H1481) were used in protein overexpression. The histidine-tagged recombinant proteins were purified from the soluble fraction (lysate) by affinity chromatography on a Ni2+ chelating column, samples were taken from the flow-through (FT) and bound proteins were eluted using 50 and 500 mM imidazole. Precipitation using trichloroacetic acid and deoxycholate was used to enhance visualization and identification of the eluted proteins by mass spectrometry. Stars denote contaminants shown in Figure 5D.
Mentions: Histidine-tagged RPA1 and RPA3 pulled down their respective RPAPs, and histidine-tagged RPAP1 and RPAP3 pulled down their respective RPAs (Figure 6). However, histidine-tagged RPA1 did not pull down RPAP3, and vice versa. This confirms that the RPAs interact specifically with their respective RPAPs, supporting our conclusions based on the failure to generate Δrpa1 Δrpap3 and Δrpa3 Δrpap1 deletion mutants. Neither RPA1 nor RPA3 pulled down RPA2, and histidine-tagged RPA2 did not pull down RPA1 or 3, or either of the RPAPs (data not shown). This supports the suggestion that the three RPAs of H. volcanii do not form a heterotrimer as observed in eukaryotes and P. furiosus, but instead form three separate ssDNA-binding factors.

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