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Specialization of an Exonuclease III family enzyme in the repair of 3' DNA lesions during base excision repair in the human pathogen Neisseria meningitidis.

Silhan J, Nagorska K, Zhao Q, Jensen K, Freemont PS, Tang CM, Baldwin GS - Nucleic Acids Res. (2011)

Bottom Line: We now reveal further functional specialization at the level of 3'-PO(4) processing for NExo.However, no such functional redundancy exists for the 3'-phosphatase activity of NExo, and the cytotoxicity of 3'-blocking lesions is reflected in the marked sensitivity of a mutant lacking NExo to oxidative stress, compared to strains deficient in other base excision repair enzymes.A histidine residue within NExo that is responsible for its lack of AP endonuclease activity is also important for its 3'-phosphatase activity, demonstrating an evolutionary trade off in enzyme function at the single amino acid level.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK.

ABSTRACT
We have previously demonstrated that the two Exonuclease III (Xth) family members present within the obligate human pathogen Neisseria meningitidis, NApe and NExo, are important for survival under conditions of oxidative stress. Of these, only NApe possesses AP endonuclease activity, while the primary function of NExo remained unclear. We now reveal further functional specialization at the level of 3'-PO(4) processing for NExo. We demonstrate that the bi-functional meningococcal glycosylases Nth and MutM can perform strand incisions at abasic sites in addition to NApe. However, no such functional redundancy exists for the 3'-phosphatase activity of NExo, and the cytotoxicity of 3'-blocking lesions is reflected in the marked sensitivity of a mutant lacking NExo to oxidative stress, compared to strains deficient in other base excision repair enzymes. A histidine residue within NExo that is responsible for its lack of AP endonuclease activity is also important for its 3'-phosphatase activity, demonstrating an evolutionary trade off in enzyme function at the single amino acid level. This specialization of two Xth enzymes for the 3'-end processing and strand-incision reactions has not previously been observed and provides a new paradigm within the prokaryotic world for separation of these critical functions during base excision repair.

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Related in: MedlinePlus

Cell extract activity. Cell lysates from wild type MC58, Δnape, Δnexo and Δnape/Δnexo strains were assayed for 3′-phosphatase, and 3′–5′ exonuclease activities. 100 nM substrate (20-PO4 or 20-OH) was incubated with 10× diluted cell extracts from indicated strains under standard phosphatase/exonuclease assay conditions for 60 min. Products were separated by 20% denaturing PAGE.
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gkr905-F7: Cell extract activity. Cell lysates from wild type MC58, Δnape, Δnexo and Δnape/Δnexo strains were assayed for 3′-phosphatase, and 3′–5′ exonuclease activities. 100 nM substrate (20-PO4 or 20-OH) was incubated with 10× diluted cell extracts from indicated strains under standard phosphatase/exonuclease assay conditions for 60 min. Products were separated by 20% denaturing PAGE.

Mentions: Examination of the genome of MC58 did not reveal any other enzyme homologies, such as poly-nucleotide kinase (PNK), that may be associated with 3′-phosphatase activities. To determine whether NExo is the only enzyme with 3′-phosphatase activity, we assayed cell extracts from MC58, MC58Δnexo, MC58Δnape and MC58Δnexo/Δnape with the 20-PO4 substrate (Figure 7). Extracts from the wild type meningococcus can clearly be seen to remove the 3′-phosphate with similar efficiency to 1 nM purified NExo, followed by rapid degradation through exonuclease activity. The NExo containing mutant extract (MC58Δnape) matched the activity of the purified NExo enzyme with efficient 3′-phosphate processing, but reduced exonuclease activity. No 3′-phosphatase activity was observed with the MC58Δnexo or MC58Δnexo/Δnape double mutant. Analysis of the 3′–5′ exonuclease activity with 20-OH substrate demonstrated that NApe and NExo account for all of the observed exonuclease activity in the cell extract. While both enzymes contribute to this, it is evident that NApe is responsible for the majority of exonuclease activity (Figure 7).Figure 7.


Specialization of an Exonuclease III family enzyme in the repair of 3' DNA lesions during base excision repair in the human pathogen Neisseria meningitidis.

Silhan J, Nagorska K, Zhao Q, Jensen K, Freemont PS, Tang CM, Baldwin GS - Nucleic Acids Res. (2011)

Cell extract activity. Cell lysates from wild type MC58, Δnape, Δnexo and Δnape/Δnexo strains were assayed for 3′-phosphatase, and 3′–5′ exonuclease activities. 100 nM substrate (20-PO4 or 20-OH) was incubated with 10× diluted cell extracts from indicated strains under standard phosphatase/exonuclease assay conditions for 60 min. Products were separated by 20% denaturing PAGE.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3300015&req=5

gkr905-F7: Cell extract activity. Cell lysates from wild type MC58, Δnape, Δnexo and Δnape/Δnexo strains were assayed for 3′-phosphatase, and 3′–5′ exonuclease activities. 100 nM substrate (20-PO4 or 20-OH) was incubated with 10× diluted cell extracts from indicated strains under standard phosphatase/exonuclease assay conditions for 60 min. Products were separated by 20% denaturing PAGE.
Mentions: Examination of the genome of MC58 did not reveal any other enzyme homologies, such as poly-nucleotide kinase (PNK), that may be associated with 3′-phosphatase activities. To determine whether NExo is the only enzyme with 3′-phosphatase activity, we assayed cell extracts from MC58, MC58Δnexo, MC58Δnape and MC58Δnexo/Δnape with the 20-PO4 substrate (Figure 7). Extracts from the wild type meningococcus can clearly be seen to remove the 3′-phosphate with similar efficiency to 1 nM purified NExo, followed by rapid degradation through exonuclease activity. The NExo containing mutant extract (MC58Δnape) matched the activity of the purified NExo enzyme with efficient 3′-phosphate processing, but reduced exonuclease activity. No 3′-phosphatase activity was observed with the MC58Δnexo or MC58Δnexo/Δnape double mutant. Analysis of the 3′–5′ exonuclease activity with 20-OH substrate demonstrated that NApe and NExo account for all of the observed exonuclease activity in the cell extract. While both enzymes contribute to this, it is evident that NApe is responsible for the majority of exonuclease activity (Figure 7).Figure 7.

Bottom Line: We now reveal further functional specialization at the level of 3'-PO(4) processing for NExo.However, no such functional redundancy exists for the 3'-phosphatase activity of NExo, and the cytotoxicity of 3'-blocking lesions is reflected in the marked sensitivity of a mutant lacking NExo to oxidative stress, compared to strains deficient in other base excision repair enzymes.A histidine residue within NExo that is responsible for its lack of AP endonuclease activity is also important for its 3'-phosphatase activity, demonstrating an evolutionary trade off in enzyme function at the single amino acid level.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Biosciences, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK.

ABSTRACT
We have previously demonstrated that the two Exonuclease III (Xth) family members present within the obligate human pathogen Neisseria meningitidis, NApe and NExo, are important for survival under conditions of oxidative stress. Of these, only NApe possesses AP endonuclease activity, while the primary function of NExo remained unclear. We now reveal further functional specialization at the level of 3'-PO(4) processing for NExo. We demonstrate that the bi-functional meningococcal glycosylases Nth and MutM can perform strand incisions at abasic sites in addition to NApe. However, no such functional redundancy exists for the 3'-phosphatase activity of NExo, and the cytotoxicity of 3'-blocking lesions is reflected in the marked sensitivity of a mutant lacking NExo to oxidative stress, compared to strains deficient in other base excision repair enzymes. A histidine residue within NExo that is responsible for its lack of AP endonuclease activity is also important for its 3'-phosphatase activity, demonstrating an evolutionary trade off in enzyme function at the single amino acid level. This specialization of two Xth enzymes for the 3'-end processing and strand-incision reactions has not previously been observed and provides a new paradigm within the prokaryotic world for separation of these critical functions during base excision repair.

Show MeSH
Related in: MedlinePlus