Abstract
High-fidelity DNA replication depends on a proofreading 3′–5′ exonuclease that is associated with the replicative DNA polymerase. The replicative DNA polymerase DnaE1 from the major pathogen Mycobacterium tuberculosis (Mtb) uses its intrinsic PHP-exonuclease that is distinct from the canonical DEDD exonucleases found in the Escherichia coli and eukaryotic replisomes. The mechanism of the PHP-exonuclease is not known. Here, we present the crystal structure of the Mtb DnaE1 polymerase. The PHP-exonuclease has a trinuclear zinc center, coordinated by nine conserved residues. Cryo-EM analysis reveals the entry path of the primer strand in the PHP-exonuclease active site. Furthermore, the PHP-exonuclease shows a striking similarity to E. coli endonuclease IV, which provides clues regarding the mechanism of action. Altogether, this work provides important insights into the PHP-exonuclease and reveals unique properties that make it an attractive target for novel anti-mycobacterial drugs.
Highlights
High-fidelity DNA replication depends on a proofreading 3′–5′ exonuclease that is associated with the replicative DNA polymerase
We have shown that the PHP domain of Mycobacterium tuberculosis (Mtb) DnaE1 is a potential target for new antibiotics as its inactivation leads to strongly decreased growth rates and renders mycobacteria sensitive to nucleotide analogs that are used in antiviral therapy
We found that the active site of the DnaE1 PHP domain shows a striking similarity to the active site of the E. coli endonuclease IV (Endo IV), an enzyme involved in base excision repair, whose mechanism has been studied in great detail[28,29,30]
Summary
On the other side of the thumb domain, ~40 Å away from the polymerase active site, is the exonuclease active site (Fig. 1a) It is located in the center of the PHP domain and consists of nine conserved residues that coordinate three metals (Fig. 2a, b). To reveal how the primer strand reaches the PHP-exonuclease active site, we used cryo-EM to determine the structure of DnaE1 bound to the β-clamp and a mismatched DNA substrate. We found that the active site of the DnaE1 PHP domain shows a striking similarity to the active site of the E. coli endonuclease IV (Endo IV), an enzyme involved in base excision repair, whose mechanism has been studied in great detail[28,29,30]
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