Limits...
Deinococcus radiodurans PriA is a Pseudohelicase.

Lopper ME, Boone J, Morrow C - PLoS ONE (2015)

Bottom Line: Here, we show that PriA from a highly radiation-resistant member of that phylum, Deinococcus radiodurans, lacks the ability to hydrolyze ATP and unwind duplex DNA, thus qualifying D. radiodurans PriA as a pseudohelicase.Despite the lack of helicase activity, D. radiodurans PriA has retained the DNA binding activity expected of a typical PriA helicase, and we present evidence for a physical interaction between D. radiodurans PriA and its cognate replicative helicase, DnaB.This suggests that PriA has retained a role in replisome reloading onto repaired DNA replication forks in D. radiodurans despite its lack of helicase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Dayton, Dayton, OH, United States of America.

ABSTRACT
Reactivation of repaired DNA replication forks in bacteria is catalyzed by PriA helicase. This broadly-conserved bacterial enzyme can remodel the structure of DNA at a repaired DNA replication fork by unwinding small portions of duplex DNA to prepare the fork for replisome reloading. While PriA's helicase activity is not strictly required for cell viability in E. coli, the sequence motifs that confer helicase activity upon PriA are well-conserved among sequenced bacterial priA genes, suggesting that PriA's duplex DNA unwinding activity confers a selective advantage upon cells. However, these helicase sequence motifs are not well-conserved among priA genes from the Deinococcus-Thermus phylum. Here, we show that PriA from a highly radiation-resistant member of that phylum, Deinococcus radiodurans, lacks the ability to hydrolyze ATP and unwind duplex DNA, thus qualifying D. radiodurans PriA as a pseudohelicase. Despite the lack of helicase activity, D. radiodurans PriA has retained the DNA binding activity expected of a typical PriA helicase, and we present evidence for a physical interaction between D. radiodurans PriA and its cognate replicative helicase, DnaB. This suggests that PriA has retained a role in replisome reloading onto repaired DNA replication forks in D. radiodurans despite its lack of helicase activity.

No MeSH data available.


Evolutionary conservation of PriA helicase motifs.Sequence logos were generated for helicase motifs I and II (the Walker A and Walker B boxes, respectively) from a multiple sequence alignment of 100 PriA helicases using the Weblogo 3 sequence logo generator [27, 28]. Each PriA sequence represents a unique genus, and 18 phyla are represented. A multiple sequence alignment of 17 members of the Deinococcus-Thermus phylum appears below each sequence logo. These sequences show significant departure from the otherwise well-conserved helicase motifs.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4504706&req=5

pone.0133419.g002: Evolutionary conservation of PriA helicase motifs.Sequence logos were generated for helicase motifs I and II (the Walker A and Walker B boxes, respectively) from a multiple sequence alignment of 100 PriA helicases using the Weblogo 3 sequence logo generator [27, 28]. Each PriA sequence represents a unique genus, and 18 phyla are represented. A multiple sequence alignment of 17 members of the Deinococcus-Thermus phylum appears below each sequence logo. These sequences show significant departure from the otherwise well-conserved helicase motifs.

Mentions: Members of the Deinococcus-Thermus phylum are notable exceptions to the evolutionary tendency to conserve PriA’s helicase motifs. These bacteria encode a PriA helicase that lacks amino acid residues that are otherwise well-conserved among known bacterial PriAs and are critical for catalyzing ATP hydrolysis and facilitating duplex DNA unwinding. In particular, the Deinococcus-Thermus phylum members have poor matches to the consensus sequences of helicase motifs I and II, also known as the Walker A and Walker B boxes, respectively (Fig 2). In E. coli PriA and other well-studied helicases, the Walker A box (also known as a phosphate-binding loop, or P-loop) is involved in ATP binding, while the Walker B box is involved in binding an active site Mg2+. Both motifs are therefore critical for helicase-catalyzed duplex DNA unwinding [16–18]. Within the Walker A box of Deinococcus-Thermus PriAs, the catalytic lysine residue in the GKT sequence of the motif has been substituted for an arginine residue (Fig 2). This amino acid substitution has been constructed and studied in E. coli PriA (the K230R PriA variant), and it has been shown to be sufficient to abolish E. coli PriA’s ATPase and helicase activities [13]. Taken together, these observations led us to hypothesize that Deinococcus-Thermus PriA enzymes would be deficient at hydrolyzing ATP and unwinding duplex DNA, effectively qualifying them as pseudohelicases–enzymes that have clearly evolved from an ancestral helicase but have lost their catalytic activity.


Deinococcus radiodurans PriA is a Pseudohelicase.

Lopper ME, Boone J, Morrow C - PLoS ONE (2015)

Evolutionary conservation of PriA helicase motifs.Sequence logos were generated for helicase motifs I and II (the Walker A and Walker B boxes, respectively) from a multiple sequence alignment of 100 PriA helicases using the Weblogo 3 sequence logo generator [27, 28]. Each PriA sequence represents a unique genus, and 18 phyla are represented. A multiple sequence alignment of 17 members of the Deinococcus-Thermus phylum appears below each sequence logo. These sequences show significant departure from the otherwise well-conserved helicase motifs.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4504706&req=5

pone.0133419.g002: Evolutionary conservation of PriA helicase motifs.Sequence logos were generated for helicase motifs I and II (the Walker A and Walker B boxes, respectively) from a multiple sequence alignment of 100 PriA helicases using the Weblogo 3 sequence logo generator [27, 28]. Each PriA sequence represents a unique genus, and 18 phyla are represented. A multiple sequence alignment of 17 members of the Deinococcus-Thermus phylum appears below each sequence logo. These sequences show significant departure from the otherwise well-conserved helicase motifs.
Mentions: Members of the Deinococcus-Thermus phylum are notable exceptions to the evolutionary tendency to conserve PriA’s helicase motifs. These bacteria encode a PriA helicase that lacks amino acid residues that are otherwise well-conserved among known bacterial PriAs and are critical for catalyzing ATP hydrolysis and facilitating duplex DNA unwinding. In particular, the Deinococcus-Thermus phylum members have poor matches to the consensus sequences of helicase motifs I and II, also known as the Walker A and Walker B boxes, respectively (Fig 2). In E. coli PriA and other well-studied helicases, the Walker A box (also known as a phosphate-binding loop, or P-loop) is involved in ATP binding, while the Walker B box is involved in binding an active site Mg2+. Both motifs are therefore critical for helicase-catalyzed duplex DNA unwinding [16–18]. Within the Walker A box of Deinococcus-Thermus PriAs, the catalytic lysine residue in the GKT sequence of the motif has been substituted for an arginine residue (Fig 2). This amino acid substitution has been constructed and studied in E. coli PriA (the K230R PriA variant), and it has been shown to be sufficient to abolish E. coli PriA’s ATPase and helicase activities [13]. Taken together, these observations led us to hypothesize that Deinococcus-Thermus PriA enzymes would be deficient at hydrolyzing ATP and unwinding duplex DNA, effectively qualifying them as pseudohelicases–enzymes that have clearly evolved from an ancestral helicase but have lost their catalytic activity.

Bottom Line: Here, we show that PriA from a highly radiation-resistant member of that phylum, Deinococcus radiodurans, lacks the ability to hydrolyze ATP and unwind duplex DNA, thus qualifying D. radiodurans PriA as a pseudohelicase.Despite the lack of helicase activity, D. radiodurans PriA has retained the DNA binding activity expected of a typical PriA helicase, and we present evidence for a physical interaction between D. radiodurans PriA and its cognate replicative helicase, DnaB.This suggests that PriA has retained a role in replisome reloading onto repaired DNA replication forks in D. radiodurans despite its lack of helicase activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Dayton, Dayton, OH, United States of America.

ABSTRACT
Reactivation of repaired DNA replication forks in bacteria is catalyzed by PriA helicase. This broadly-conserved bacterial enzyme can remodel the structure of DNA at a repaired DNA replication fork by unwinding small portions of duplex DNA to prepare the fork for replisome reloading. While PriA's helicase activity is not strictly required for cell viability in E. coli, the sequence motifs that confer helicase activity upon PriA are well-conserved among sequenced bacterial priA genes, suggesting that PriA's duplex DNA unwinding activity confers a selective advantage upon cells. However, these helicase sequence motifs are not well-conserved among priA genes from the Deinococcus-Thermus phylum. Here, we show that PriA from a highly radiation-resistant member of that phylum, Deinococcus radiodurans, lacks the ability to hydrolyze ATP and unwind duplex DNA, thus qualifying D. radiodurans PriA as a pseudohelicase. Despite the lack of helicase activity, D. radiodurans PriA has retained the DNA binding activity expected of a typical PriA helicase, and we present evidence for a physical interaction between D. radiodurans PriA and its cognate replicative helicase, DnaB. This suggests that PriA has retained a role in replisome reloading onto repaired DNA replication forks in D. radiodurans despite its lack of helicase activity.

No MeSH data available.