Editing of misaligned 3'-termini by an intrinsic 3'-5' exonuclease activity residing in the PHP domain of a family X DNA polymerase

Abstract

Bacillus subtilis gene yshC encodes a family X DNA polymerase (PolXBs), whose biochemical features suggest that it plays a role during DNA repair processes. Here, we show that, in addition to the polymerization activity, PolXBs possesses an intrinsic 3′–5′ exonuclease activity specialized in resecting unannealed 3′-termini in a gapped DNA substrate. Biochemical analysis of a PolXBs deletion mutant lacking the C-terminal polymerase histidinol phosphatase (PHP) domain, present in most of the bacterial/archaeal PolXs, as well as of this separately expressed protein region, allow us to state that the 3′–5′ exonuclease activity of PolXBs resides in its PHP domain. Furthermore, site-directed mutagenesis of PolXBs His339 and His341 residues, evolutionary conserved in the PHP superfamily members, demonstrated that the predicted metal binding site is directly involved in catalysis of the exonucleolytic reaction. The implications of the unannealed 3′-termini resection by the 3′–5′ exonuclease activity of PolXBs in the DNA repair context are discussed.

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

“…1B shows that, in both cases, the maximal activity was reached with fraction 12, coincident with the mass peak. The 3′-5′-exonuclease activity exhibited a distributive pattern, as described (12), giving rise to 26-33-mer degradation intermediates. The absence of the 18-mer product rules out the 3′-5′-exonuclease activity as the one responsible for the generation of the product obtained with the THF-containing substrate, confirming the specificity of the nicking activity for an AP site.…”

“…Conversely, PolX Bs possesses a metal-dependent nicking activity on AP sites because Mg 2þ and Mn 2þ cations promoted the enzyme to cleave at the THF position, giving rise to the 18-mer product. The shorter bands observed with Mn 2þ result from the 3′-5′ exonucleolytic degradation of the incised AP site by PolX Bs (12), showing that the single-stranded break introduced by the polymerase is prone to further exonucleolysis. These results contrast with the absence of internal cleavage of a nondamaged DNA that is in fact degraded progressively from the 3′ ends by the intrinsic 3′-5′-exonuclease activity of PolX Bs , as previously reported (12) (see also Fig.…”

“…To map the PolX Bs domain responsible for the AP-endonuclease activity, the presence of the latter was analyzed in the separately expressed and purified polymerization and PHP domains (12). Fig.…”

“…The 10-fold reduction of the catalytic efficiency exhibited by this domain respect to the complete enzyme suggests that proficient endonucleolysis would require the assistance of the polymerization domain. Multiple sequence alignment of the PHP domain of bacterial/ archaeal PolXs permitted the identification of highly conserved residues grouped in four motifs, assembling a catalytic core that would coordinate the metal ions responsible for the 3′-5′-exonuclease activity (12). To ascertain the involvement of the PHP active site in supporting catalysis of both, the 3′-5′-exonuclease and AP-endonuclease activities, mutants D346A (motif I), H371A (motif II), E410A and H437A (motif III), H465A, and D526A and H528A (motif IV), at the corresponding PolX Bs residues, were obtained (see Materials and Methods).…”

“…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)(12)(13)(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 pathways during the B. subtilis life cycle, as it has been suggested recently for other bacterial PolXs (10,15).…”