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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) Map of rpa1 operon indicating location of Δrpa1, Δrpap1, and Δrpa1 operon deletions, as well as the BspEI and NruI sites, and probes used to verify the deletions. (B) Southern blot of genomic DNA cut with BspEI and probed with flanking regions of rpa1 and rpap1, as shown in (A), to indicate deletion of rpa1 and rpap1, respectively. (C) Southern blot of genomic DNA cut with NruI and probed with flanking regions of rpa1 operon (rpa1 op.), as shown in (A), to indicate deletion of the rpa1 operon.
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Figure 2: (A) Map of rpa1 operon indicating location of Δrpa1, Δrpap1, and Δrpa1 operon deletions, as well as the BspEI and NruI sites, and probes used to verify the deletions. (B) Southern blot of genomic DNA cut with BspEI and probed with flanking regions of rpa1 and rpap1, as shown in (A), to indicate deletion of rpa1 and rpap1, respectively. (C) Southern blot of genomic DNA cut with NruI and probed with flanking regions of rpa1 operon (rpa1 op.), as shown in (A), to indicate deletion of the rpa1 operon.

Mentions: In eukaryotes, specifically Saccharomyces cerevisiae, all three RPA subunits have been shown to be essential for cell survival (Brill and Stillman, 1991). Work by Skowyra and MacNeill (2012) has shown that H. volcaniirpa2 is essential, which is in agreement with our fruitless attempts to delete rpa2 (data not shown). To examine if the other rpa genes of H. volcanii are also essential, genomic deletions of rpa1 and rpa3 were generated using the counter selective pop-in/pop-out method (Allers et al., 2004). To generate the deletion constructs by PCR, rpa1 and rpa3 operons were first isolated from wild-type (WT) H. volcanii using native BspEI and EcoRI/NotI restriction sites, respectively, to generate genomic libraries. These were then screened for the presence of the rpa1 and rpa3 operons, individually, using colony hybridization. The isolated plasmids, pTA937 (rpa1 operon) and pTA884 (rpa3 operon) were confirmed by DNA sequencing. Deletion constructs for rpa1 and rpa3 were designed to avoid polar effects on the expression of the downstream rpap genes by maintaining the reading frame. Genomic deletions of both rpa1 and rpa3 (trpA-marked) were successful, generating strains H1217 and H1244, respectively (Figures 2 and 3, respectively). The ability to delete both rpa1 and rpa3 with relative ease, but not rpa2, indicates that the cellular requirement for each RPA is not equal, making it unlikely that they function collectively.


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) Map of rpa1 operon indicating location of Δrpa1, Δrpap1, and Δrpa1 operon deletions, as well as the BspEI and NruI sites, and probes used to verify the deletions. (B) Southern blot of genomic DNA cut with BspEI and probed with flanking regions of rpa1 and rpap1, as shown in (A), to indicate deletion of rpa1 and rpap1, respectively. (C) Southern blot of genomic DNA cut with NruI and probed with flanking regions of rpa1 operon (rpa1 op.), as shown in (A), to indicate deletion of the rpa1 operon.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: (A) Map of rpa1 operon indicating location of Δrpa1, Δrpap1, and Δrpa1 operon deletions, as well as the BspEI and NruI sites, and probes used to verify the deletions. (B) Southern blot of genomic DNA cut with BspEI and probed with flanking regions of rpa1 and rpap1, as shown in (A), to indicate deletion of rpa1 and rpap1, respectively. (C) Southern blot of genomic DNA cut with NruI and probed with flanking regions of rpa1 operon (rpa1 op.), as shown in (A), to indicate deletion of the rpa1 operon.
Mentions: In eukaryotes, specifically Saccharomyces cerevisiae, all three RPA subunits have been shown to be essential for cell survival (Brill and Stillman, 1991). Work by Skowyra and MacNeill (2012) has shown that H. volcaniirpa2 is essential, which is in agreement with our fruitless attempts to delete rpa2 (data not shown). To examine if the other rpa genes of H. volcanii are also essential, genomic deletions of rpa1 and rpa3 were generated using the counter selective pop-in/pop-out method (Allers et al., 2004). To generate the deletion constructs by PCR, rpa1 and rpa3 operons were first isolated from wild-type (WT) H. volcanii using native BspEI and EcoRI/NotI restriction sites, respectively, to generate genomic libraries. These were then screened for the presence of the rpa1 and rpa3 operons, individually, using colony hybridization. The isolated plasmids, pTA937 (rpa1 operon) and pTA884 (rpa3 operon) were confirmed by DNA sequencing. Deletion constructs for rpa1 and rpa3 were designed to avoid polar effects on the expression of the downstream rpap genes by maintaining the reading frame. Genomic deletions of both rpa1 and rpa3 (trpA-marked) were successful, generating strains H1217 and H1244, respectively (Figures 2 and 3, respectively). The ability to delete both rpa1 and rpa3 with relative ease, but not rpa2, indicates that the cellular requirement for each RPA is not equal, making it unlikely that they function collectively.

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