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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

Operon and domain structures of H. volcanii single-stranded DNA-binding proteins. Genes for RPA1 and RPA3 are in operons with genes for RPA-associated proteins, RPAP1 and RPAP3, respectively. The gene for RPA1 phosphoesterase (RPE) is present in the rpa1 operon. Domains (not to scale) comprising OB-folds, zinc fingers and a phosphoesterase motif are shown.
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Figure 1: Operon and domain structures of H. volcanii single-stranded DNA-binding proteins. Genes for RPA1 and RPA3 are in operons with genes for RPA-associated proteins, RPAP1 and RPAP3, respectively. The gene for RPA1 phosphoesterase (RPE) is present in the rpa1 operon. Domains (not to scale) comprising OB-folds, zinc fingers and a phosphoesterase motif are shown.

Mentions: Haloferax volcanii encodes three RPA genes rpa1, rpa2, and rpa3 (Hartman et al., 2010). Recent studies have shown RPA2 to be essential while RPA1 and RPA3 are not (Skowyra and MacNeill, 2012). Note that these authors used the nomenclature rpaA1, A2, B1, B2, B3, and C to refer to rpa3, rpap3, rpa1, rpap1, rpe, and rpa2, respectively, while we have chosen to maintain the official nomenclature as described in Table 4 of the H. volcanii genome paper (Hartman et al., 2010). Both rpa1 and rpa3 are in operons with other genes; rpa1 is in an operon with genes encoding an OB-fold containing protein (hereby designated RPA-associated protein or RPAP) and a calcineurin-like phosphoesterase, while only one OB-fold rpa-associated protein (rpap) gene is present in the rpa3 operon (Figure 1). The presence of an rpap gene in the same operon as rpa can be found in other euryarchaeota, including Halobacterium marismortui, Halobacterium salinarum, and Natronomonas pharaonis, as well as in M. mazei and M. barkeri. The rpap gene has been assigned to the cluster of orthologous groups (COG) 3390 (Berthon et al., 2008).


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

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

Operon and domain structures of H. volcanii single-stranded DNA-binding proteins. Genes for RPA1 and RPA3 are in operons with genes for RPA-associated proteins, RPAP1 and RPAP3, respectively. The gene for RPA1 phosphoesterase (RPE) is present in the rpa1 operon. Domains (not to scale) comprising OB-folds, zinc fingers and a phosphoesterase motif are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Operon and domain structures of H. volcanii single-stranded DNA-binding proteins. Genes for RPA1 and RPA3 are in operons with genes for RPA-associated proteins, RPAP1 and RPAP3, respectively. The gene for RPA1 phosphoesterase (RPE) is present in the rpa1 operon. Domains (not to scale) comprising OB-folds, zinc fingers and a phosphoesterase motif are shown.
Mentions: Haloferax volcanii encodes three RPA genes rpa1, rpa2, and rpa3 (Hartman et al., 2010). Recent studies have shown RPA2 to be essential while RPA1 and RPA3 are not (Skowyra and MacNeill, 2012). Note that these authors used the nomenclature rpaA1, A2, B1, B2, B3, and C to refer to rpa3, rpap3, rpa1, rpap1, rpe, and rpa2, respectively, while we have chosen to maintain the official nomenclature as described in Table 4 of the H. volcanii genome paper (Hartman et al., 2010). Both rpa1 and rpa3 are in operons with other genes; rpa1 is in an operon with genes encoding an OB-fold containing protein (hereby designated RPA-associated protein or RPAP) and a calcineurin-like phosphoesterase, while only one OB-fold rpa-associated protein (rpap) gene is present in the rpa3 operon (Figure 1). The presence of an rpap gene in the same operon as rpa can be found in other euryarchaeota, including Halobacterium marismortui, Halobacterium salinarum, and Natronomonas pharaonis, as well as in M. mazei and M. barkeri. The rpap gene has been assigned to the cluster of orthologous groups (COG) 3390 (Berthon et al., 2008).

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