Identification of an Intrinsic 5′-Deoxyribose-5-phosphate Lyase Activity in Human DNA Polymerase λ: A POSSIBLE ROLE IN BASE EXCISION REPAIR

Katarzyna Bebenek,Miguel Garcı́A-Dı́Az,Thomas A Kunkel,Luis Blanco

doi:10.1074/jbc.m106336200

Abstract

Base excision repair (BER) is a major repair pathway in eukaryotic cells responsible for repair of lesions that give rise to abasic (AP) sites in DNA. Pivotal to this process is the 5'-deoxyribose-5-phosphate lyase (dRP lyase) activity of DNA polymerase beta (Pol beta). DNA polymerase lambda (Pol lambda) is a recently identified eukaryotic DNA polymerase that is homologous to Pol beta. We show here that human Pol lambda exhibits dRP lyase, but not AP lyase, activity in vitro and that this activity is consistent with a beta-elimination mechanism. Accordingly, a single amino acid substitution (K310A) eliminated more than 90% of the wild-type dRP lyase activity, thus suggesting that Lys(310) of Pol lambda is the main nucleophile involved in the reaction. The dRP lyase activity of Pol lambda, in coordination with its polymerization activity, efficiently repaired uracil-containing DNA in an in vitro reconstituted BER reaction. These results suggest that Pol lambda may participate in "single-nucleotide" base excision repair in mammalian cells.

Highlights

Aspergillus fumigatus is a leading cause of invasive fungal infections
Clinical isolates of A. fumigatus obtained from patients with chronic pulmonary aspergillosis who failed echinocandin therapy were shown to have elevated Minimum effective concentrations (MECs) for both CAS and micafungin (MFG) (Table 1)
DNA sequence analysis revealed no mutations in the fks1 gene open reading frame or promoter, suggesting that the mechanism of echinocandin resistance in these strains was independent of the established FKS mechanism of well-characterized Candida species [4] and known to exist in Aspergillus fumigatus [6, 7]

Summary

Introduction

Aspergillus fumigatus is a leading cause of invasive fungal infections. Resistance to first-line triazole antifungals has led to therapy with echinocandin drugs. Echinocandin resistance is known to arise from amino acid substitutions in ␤-(1,3)-D-glucan synthase encoded by the fks gene These clinical isolates did not contain mutations in fks, indicating an undefined resistance mechanism. As the number of Aspergillus-infected patients treated with echinocandins is rising, clinical observations of drug resistance are increasing, indicating an emerging global health threat. Mechanisms of echinocandin resistance have been extensively studied in yeasts of the Candida genus In these organisms, clinical resistance to echinocandins arises via mutations in the hot spot regions of FKS genes which encode the cell wall biosynthetic enzyme ␤-(1,3)-D-glucan synthase [5]. It was reported that upregulation of glucan synthase may result in reduced clinical drug response [9] These observations point to the clinical relevance of fks mutation-independent mechanisms for echinocandin resistance in A. fumigatus

Methods

Results

Conclusion