Intrinsic apurinic/apyrimidinic (AP) endonuclease activity enables Bacillus subtilis DNA polymerase X to recognize, incise, and further repair abasic sites

Abstract

The N-glycosidic bond can be hydrolyzed spontaneously or by glycosylases during removal of damaged bases by the base excision repair pathway, leading to the formation of highly mutagenic apurinic/apyrimidinic (AP) sites. Organisms encode for evolutionarily conserved repair machinery, including specific AP endonucleases that cleave the DNA backbone 5′ to the AP site to prime further DNA repair synthesis. We report on the DNA polymerase X from the bacterium Bacillus subtilis (PolX Bs ) that, along with polymerization and 3′-5′-exonuclease activities, possesses an intrinsic AP-endonuclease activity. Both, AP-endonuclease and 3′-5′-exonuclease activities are genetically linked and governed by the same metal ligands located at the C-terminal polymerase and histidinol phosphatase domain of the polymerase. The different catalytic functions of PolX Bs enable it to perform recognition and incision at an AP site and further restoration (repair) of the original nucleotide in a standalone AP-endonuclease-independent way. apurinic/apyrimidinic-lyase | site-directed mutagenesis G enomes are continuously insulted by exogenous and endogenous genotoxic agents, as ionizing radiation, drugs, and (by)products of normal cellular metabolism that generate reactive oxygen species (ROS) leading to mainly nonbulky DNA lesions (1). Base excision repair (BER) is the major pathway involved in the removal of this type of damage, and its importance for cell survival is reflected by its conservation from bacteria to eukaryotes (2). During the first steps of BER, highly mutagenic apurinic/apyrimidinic (AP) intermediates are produced as a result of hydrolytic cleavage of the altered base-sugar bond by mono-(class II) and/or bifunctional (class I) DNA N-glycosylases (ref. 3 and references therein), or from spontaneous DNA base loss, causing replication and transcription inhibition if left unrepaired (4, 5). AP endonucleases play a crucial role in BER because they recognize the abasic residue and hydrolyze the phosphodiester bond 5′ to the AP site, leaving a gapped DNA intermediate with an extendable 3′-OH end (ref. 2 and references therein).Members of the family X of DNA polymerases (hereafter, PolX) are widely spread in nature from virus to humans, being involved in the DNA synthesis step during BER and DNA double-strand break repair by virtue of a common Polβ-like core adapted to fill the gapped DNA intermediates very proficiently (6-9).PolX Bs (570-aa long) is a prototypic bacterial/archaeal PolX member from Bacillus subtilis with a N-terminal Polβ-like core (residues 1-317) responsible for catalysis of DNA polymerization (10), and a C-terminal polymerase and histidinol phosphatase (PHP) domain (residues 333-570) containing highly conserved residues that catalyze a Mn 2þ -dependent 3′-5′-exonuclease activity (11-14), which shows a preferential processing of unannealed 3′ termini (12). Due to this fact and to its adaptation to perform filling of small gaps (10), PolX Bs was proposed to play a potential role in the DNA synthesis step of repair p...

DNA Repair and Genome Maintenance in Bacillus subtilis

“…Polymerase activity is increased by the presence of a phosphate group on the 5= terminus 1 to 5 nucleotides downstream of a 3=-OH (14). In addition, it was shown that PolX is able to nick a DNA substrate containing a nucleotide analog that mimics an AP site (15). The combination of AP endonuclease activity and the preference of its polymerase activity for small gapped substrates strongly suggests that PolX may be the primary polymerase that repairs small lesions, such as those produced by BER.…”

“…The combination of AP endonuclease activity and the preference of its polymerase activity for small gapped substrates strongly suggests that PolX may be the primary polymerase that repairs small lesions, such as those produced by BER. Furthermore, PolX possesses a 3=-5= exonuclease activity which was shown to process AP sites that were cleaved on the 3= side by an AP lyase (15). Lyase cleavage produces a 3=-phospho-␣,␤-unsaturated aldehyde (3=-PUA), a group that blocks replication unless it is processed further (15).…”

“…Furthermore, PolX possesses a 3=-5= exonuclease activity which was shown to process AP sites that were cleaved on the 3= side by an AP lyase (15). Lyase cleavage produces a 3=-phospho-␣,␤-unsaturated aldehyde (3=-PUA), a group that blocks replication unless it is processed further (15). Therefore, PolX 3=-5= exonuclease activity will process 3=-PUA by hydrolyzing the phosphodiester bond, leaving a 3=-OH from which PolX can extend (15).…”

“…Lyase cleavage produces a 3=-phospho-␣,␤-unsaturated aldehyde (3=-PUA), a group that blocks replication unless it is processed further (15). Therefore, PolX 3=-5= exonuclease activity will process 3=-PUA by hydrolyzing the phosphodiester bond, leaving a 3=-OH from which PolX can extend (15). Interestingly, the exonuclease and AP endonuclease activities could not be separated by mutational analysis, and the presence of an AP site appears to inhibit the exonuclease activity of PolX, thereby favoring the AP endonuclease activity when an AP site is present (15).…”

Refining the Genetic Alphabet: A Late-Period Selection Pressure?

“…maintaining genomic stability combined with proper replication (Banos et al, 2010). Yet, particularly advantageous to the evolution of BER was the recruitment of a class of enzymes known as glycosylases that specifically undertake the task of finding and removing damaged bases as a form of preventive measure to ensure the ''health'' of a cells' genome (O'Brien, 2006).…”