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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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The intermediates of base excision repair. The different 3′-moieties generated during base excision repair. 3′-OH products are competent for further repair by DNA polymerase and ligase, while 3′–3′-Ald and 3′-PO4 cannot be extended by DNA polymerase and are thus blocking lesions.
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gkr905-F1: The intermediates of base excision repair. The different 3′-moieties generated during base excision repair. 3′-OH products are competent for further repair by DNA polymerase and ligase, while 3′–3′-Ald and 3′-PO4 cannot be extended by DNA polymerase and are thus blocking lesions.

Mentions: DNA glycosylases are responsible for recognizing and removing damaged DNA bases, and those involved in oxidative damage frequently remove multiple DNA lesions that can arise following oxidative damage (3). Glycosylases can also be bi-functional and incise the DNA backbone following removal of the base. The chemistry of these backbone incisions proceeds via an AP lyase activity in which base expulsion results from nucloephlilic attack of the 1′ of the ribose sugar, typically by the N-terminal amine or a lysine residue in the enzyme, with subsequent ring opening and strand cleavage occurring either via β-elimination to yield a 3′-unsaturated aldehyde (3′-Ald; Figure 1) or β-δ elimination leaving a 3′-phosphate (3′-PO4; Figure 1) (4,5). These 3′-lesions cannot be repaired by a DNA polymerase and are therefore referred to as 3′-blocking lesions (2,6,7); such 3′-blocking lesions can also arise directly from oxidative damage leading to 3′-PO4 and 3′-phosphoglycolate lesions (8). Further processing of these 3′-lesions typically occurs via the Xth or Nfo AP endonucleases to yield a 3′-OH that is competent for extension by a DNA polymerase (2,8).Figure 1.


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)

The intermediates of base excision repair. The different 3′-moieties generated during base excision repair. 3′-OH products are competent for further repair by DNA polymerase and ligase, while 3′–3′-Ald and 3′-PO4 cannot be extended by DNA polymerase and are thus blocking lesions.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr905-F1: The intermediates of base excision repair. The different 3′-moieties generated during base excision repair. 3′-OH products are competent for further repair by DNA polymerase and ligase, while 3′–3′-Ald and 3′-PO4 cannot be extended by DNA polymerase and are thus blocking lesions.
Mentions: DNA glycosylases are responsible for recognizing and removing damaged DNA bases, and those involved in oxidative damage frequently remove multiple DNA lesions that can arise following oxidative damage (3). Glycosylases can also be bi-functional and incise the DNA backbone following removal of the base. The chemistry of these backbone incisions proceeds via an AP lyase activity in which base expulsion results from nucloephlilic attack of the 1′ of the ribose sugar, typically by the N-terminal amine or a lysine residue in the enzyme, with subsequent ring opening and strand cleavage occurring either via β-elimination to yield a 3′-unsaturated aldehyde (3′-Ald; Figure 1) or β-δ elimination leaving a 3′-phosphate (3′-PO4; Figure 1) (4,5). These 3′-lesions cannot be repaired by a DNA polymerase and are therefore referred to as 3′-blocking lesions (2,6,7); such 3′-blocking lesions can also arise directly from oxidative damage leading to 3′-PO4 and 3′-phosphoglycolate lesions (8). Further processing of these 3′-lesions typically occurs via the Xth or Nfo AP endonucleases to yield a 3′-OH that is competent for extension by a DNA polymerase (2,8).Figure 1.

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