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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) Protein sequence alignment of C-terminus of Cdc48d from selected species of haloarchaea (Hvo, H. volcanii; Hsa, Halobacterium salinarum; Hma, Haloarcula marismortui; Hwa, Haloquadratum walsbyi; Hla, Halorubrum lacusprofundi; Nph, N. pharaonis; Hvo-Ct, H. volcanii C-terminal truncation Cdc48d-Ct). Histidine residues are indicated by a black background. (B) Colony hybridization of 5-FOA-resistant clones of H. volcanii H1333, after pop-in/pop-out gene replacement with pTA1294. H. salinarum cdc48d sequences (Hsa-cdc48d) were used as a probe, clones failing to hybridize therefore carry the truncated H. volcanii cdc48d-Ct allele present in pTA1294. (C) Verification of truncated cdc48d-Ct allele in H1424 by PCR (488 bp product), with primers specific to either H. volcanii or H. salinarum genes. H1209 genomic DNA was used as a control for wild-type H. volcanii cdc48d (563 bp product), and H1333 was used as a control for H. salinarum cdc48d (560 bp product). (D)H. volcanii strains H1209 and H1424 containing empty vector pTA963 (Allers et al., 2010) were used in mock protein overexpression. Histidine-rich cellular proteins were purified from the soluble fraction (lysate) by affinity chromatography on a Ni2+ chelating column, samples were taken from the flow-through (flow) 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. Cdc48d (HVO_1907) eluted from cell extracts of H1209 but not from H1424 (cdc48d-Ct).
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Figure 5: (A) Protein sequence alignment of C-terminus of Cdc48d from selected species of haloarchaea (Hvo, H. volcanii; Hsa, Halobacterium salinarum; Hma, Haloarcula marismortui; Hwa, Haloquadratum walsbyi; Hla, Halorubrum lacusprofundi; Nph, N. pharaonis; Hvo-Ct, H. volcanii C-terminal truncation Cdc48d-Ct). Histidine residues are indicated by a black background. (B) Colony hybridization of 5-FOA-resistant clones of H. volcanii H1333, after pop-in/pop-out gene replacement with pTA1294. H. salinarum cdc48d sequences (Hsa-cdc48d) were used as a probe, clones failing to hybridize therefore carry the truncated H. volcanii cdc48d-Ct allele present in pTA1294. (C) Verification of truncated cdc48d-Ct allele in H1424 by PCR (488 bp product), with primers specific to either H. volcanii or H. salinarum genes. H1209 genomic DNA was used as a control for wild-type H. volcanii cdc48d (563 bp product), and H1333 was used as a control for H. salinarum cdc48d (560 bp product). (D)H. volcanii strains H1209 and H1424 containing empty vector pTA963 (Allers et al., 2010) were used in mock protein overexpression. Histidine-rich cellular proteins were purified from the soluble fraction (lysate) by affinity chromatography on a Ni2+ chelating column, samples were taken from the flow-through (flow) 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. Cdc48d (HVO_1907) eluted from cell extracts of H1209 but not from H1424 (cdc48d-Ct).

Mentions: Oligonucleotides.


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) Protein sequence alignment of C-terminus of Cdc48d from selected species of haloarchaea (Hvo, H. volcanii; Hsa, Halobacterium salinarum; Hma, Haloarcula marismortui; Hwa, Haloquadratum walsbyi; Hla, Halorubrum lacusprofundi; Nph, N. pharaonis; Hvo-Ct, H. volcanii C-terminal truncation Cdc48d-Ct). Histidine residues are indicated by a black background. (B) Colony hybridization of 5-FOA-resistant clones of H. volcanii H1333, after pop-in/pop-out gene replacement with pTA1294. H. salinarum cdc48d sequences (Hsa-cdc48d) were used as a probe, clones failing to hybridize therefore carry the truncated H. volcanii cdc48d-Ct allele present in pTA1294. (C) Verification of truncated cdc48d-Ct allele in H1424 by PCR (488 bp product), with primers specific to either H. volcanii or H. salinarum genes. H1209 genomic DNA was used as a control for wild-type H. volcanii cdc48d (563 bp product), and H1333 was used as a control for H. salinarum cdc48d (560 bp product). (D)H. volcanii strains H1209 and H1424 containing empty vector pTA963 (Allers et al., 2010) were used in mock protein overexpression. Histidine-rich cellular proteins were purified from the soluble fraction (lysate) by affinity chromatography on a Ni2+ chelating column, samples were taken from the flow-through (flow) 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. Cdc48d (HVO_1907) eluted from cell extracts of H1209 but not from H1424 (cdc48d-Ct).
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Figure 5: (A) Protein sequence alignment of C-terminus of Cdc48d from selected species of haloarchaea (Hvo, H. volcanii; Hsa, Halobacterium salinarum; Hma, Haloarcula marismortui; Hwa, Haloquadratum walsbyi; Hla, Halorubrum lacusprofundi; Nph, N. pharaonis; Hvo-Ct, H. volcanii C-terminal truncation Cdc48d-Ct). Histidine residues are indicated by a black background. (B) Colony hybridization of 5-FOA-resistant clones of H. volcanii H1333, after pop-in/pop-out gene replacement with pTA1294. H. salinarum cdc48d sequences (Hsa-cdc48d) were used as a probe, clones failing to hybridize therefore carry the truncated H. volcanii cdc48d-Ct allele present in pTA1294. (C) Verification of truncated cdc48d-Ct allele in H1424 by PCR (488 bp product), with primers specific to either H. volcanii or H. salinarum genes. H1209 genomic DNA was used as a control for wild-type H. volcanii cdc48d (563 bp product), and H1333 was used as a control for H. salinarum cdc48d (560 bp product). (D)H. volcanii strains H1209 and H1424 containing empty vector pTA963 (Allers et al., 2010) were used in mock protein overexpression. Histidine-rich cellular proteins were purified from the soluble fraction (lysate) by affinity chromatography on a Ni2+ chelating column, samples were taken from the flow-through (flow) 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. Cdc48d (HVO_1907) eluted from cell extracts of H1209 but not from H1424 (cdc48d-Ct).
Mentions: Oligonucleotides.

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