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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 rpa3 operon indicating location of Δrpa3, Δrpap3, and Δrpa3 operon deletions, as well as the AscI and StuI sites and the probe used to verify the deletions. The size of this fragment in the wild-type (H195) is 8 kb. (B) Southern blot of genomic DNA cut with AscI and StuI, and probed with flanking regions of rpa3 operon (rpa3 op.), as shown in (A), indicates failure to generate Δrpa1 Δrpap3 mutant as bands of the expected size for deletion are not seen (3.4 and 4.4 kb). (C) Southern blot of genomic DNA cut with AscI and StuI, and probed with flanking regions of rpa3 op., as shown in (A), indicates failure to generate Δrpap1 Δrpa3 mutant as bands of the expected size for deletion are not seen (2.8 and 5 kb). (D) Southern blot of genomic DNA cut with AscI and StuI, and probed with flanking regions of rpa3 op., as shown in (A) indicates failure to generate Δrpa1 operon Δrpa3 operon mutant as bands of the expected size for deletion are not seen (2.8 and 3.4 kb).
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FA1: (A) Map of rpa3 operon indicating location of Δrpa3, Δrpap3, and Δrpa3 operon deletions, as well as the AscI and StuI sites and the probe used to verify the deletions. The size of this fragment in the wild-type (H195) is 8 kb. (B) Southern blot of genomic DNA cut with AscI and StuI, and probed with flanking regions of rpa3 operon (rpa3 op.), as shown in (A), indicates failure to generate Δrpa1 Δrpap3 mutant as bands of the expected size for deletion are not seen (3.4 and 4.4 kb). (C) Southern blot of genomic DNA cut with AscI and StuI, and probed with flanking regions of rpa3 op., as shown in (A), indicates failure to generate Δrpap1 Δrpa3 mutant as bands of the expected size for deletion are not seen (2.8 and 5 kb). (D) Southern blot of genomic DNA cut with AscI and StuI, and probed with flanking regions of rpa3 op., as shown in (A) indicates failure to generate Δrpa1 operon Δrpa3 operon mutant as bands of the expected size for deletion are not seen (2.8 and 3.4 kb).

Mentions: In order to test for redundancy between the two RPAs, an attempt was made to generate a double Δrpa1 operon Δrpa3 operon deletion. This involved constructing the strain H1282, which contained the pop-in of a trpA-marked Δrpa3 operon construct (pTA1207) in an unmarked Δrpa1 operon background (H1246). An episomal plasmid (pTA1265), marked with pyrE2 and providing in trans expression of the rpa1 operon was used for complementation during the pop-out step (note that this episomal plasmid is lost during counter-selection with 5-FOA). Neither of the two pop-outs generated from this strain (H1390) yielded the desired Δrpa1 operon Δrpa3 operon mutant (see Figure A1 in Appendix). This indicates that the cell requires either RPA1 or RPA3 (and/or their respective RPAPs) for survival.


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 rpa3 operon indicating location of Δrpa3, Δrpap3, and Δrpa3 operon deletions, as well as the AscI and StuI sites and the probe used to verify the deletions. The size of this fragment in the wild-type (H195) is 8 kb. (B) Southern blot of genomic DNA cut with AscI and StuI, and probed with flanking regions of rpa3 operon (rpa3 op.), as shown in (A), indicates failure to generate Δrpa1 Δrpap3 mutant as bands of the expected size for deletion are not seen (3.4 and 4.4 kb). (C) Southern blot of genomic DNA cut with AscI and StuI, and probed with flanking regions of rpa3 op., as shown in (A), indicates failure to generate Δrpap1 Δrpa3 mutant as bands of the expected size for deletion are not seen (2.8 and 5 kb). (D) Southern blot of genomic DNA cut with AscI and StuI, and probed with flanking regions of rpa3 op., as shown in (A) indicates failure to generate Δrpa1 operon Δrpa3 operon mutant as bands of the expected size for deletion are not seen (2.8 and 3.4 kb).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

FA1: (A) Map of rpa3 operon indicating location of Δrpa3, Δrpap3, and Δrpa3 operon deletions, as well as the AscI and StuI sites and the probe used to verify the deletions. The size of this fragment in the wild-type (H195) is 8 kb. (B) Southern blot of genomic DNA cut with AscI and StuI, and probed with flanking regions of rpa3 operon (rpa3 op.), as shown in (A), indicates failure to generate Δrpa1 Δrpap3 mutant as bands of the expected size for deletion are not seen (3.4 and 4.4 kb). (C) Southern blot of genomic DNA cut with AscI and StuI, and probed with flanking regions of rpa3 op., as shown in (A), indicates failure to generate Δrpap1 Δrpa3 mutant as bands of the expected size for deletion are not seen (2.8 and 5 kb). (D) Southern blot of genomic DNA cut with AscI and StuI, and probed with flanking regions of rpa3 op., as shown in (A) indicates failure to generate Δrpa1 operon Δrpa3 operon mutant as bands of the expected size for deletion are not seen (2.8 and 3.4 kb).
Mentions: In order to test for redundancy between the two RPAs, an attempt was made to generate a double Δrpa1 operon Δrpa3 operon deletion. This involved constructing the strain H1282, which contained the pop-in of a trpA-marked Δrpa3 operon construct (pTA1207) in an unmarked Δrpa1 operon background (H1246). An episomal plasmid (pTA1265), marked with pyrE2 and providing in trans expression of the rpa1 operon was used for complementation during the pop-out step (note that this episomal plasmid is lost during counter-selection with 5-FOA). Neither of the two pop-outs generated from this strain (H1390) yielded the desired Δrpa1 operon Δrpa3 operon mutant (see Figure A1 in Appendix). This indicates that the cell requires either RPA1 or RPA3 (and/or their respective RPAPs) for survival.

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