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Bacterial DNA repair genes and their eukaryotic homologues: 1. Mutations in genes involved in base excision repair (BER) and DNA-end processors and their implication in mutagenesis and human disease

100%

Krwawicz J. , Arczewska K. D. , Speina E. , Maciejewska A. , Grzesiuk E.

2007

tom 54

nr 3

413-434

Base excision repair (BER) pathway executed by a complex network of proteins is the major system responsible for the removal of damaged DNA bases and repair of DNA single strand breaks (SSBs) generated by environmental agents, such as certain cancer therapies, or arising spontaneously during cellular metabolism. Both modified DNA bases and SSBs with ends other than 3'-OH and 5'-P are repaired either by replacement of a single or of more nucleotides in the processes called short-patch BER (SP-BER) or long-patch BER (LP-BER), respectively. In contrast to Escherichia coli cells, in human ones, the two BER sub-pathways are operated by different sets of proteins. In this review the selection between SP- and LP-BER and mutations in BER and end-processors genes and their contribution to bacterial mutagenesis and human diseases are considered.

Recombinant thermostable AP exonuclease from Thermoanaerobacter tengcongensis: cloning, expression, purification, properties and PCR application

100%

Dabrowski S. , Brillowska-Dabrowska A. , Ahring B. K.

tom 62

nr 2

Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity, especially at high temperatures. The gene encoding a homologue of AP exonuclease was cloned from the thermophilic anaerobic bacterium Thermoanaerobacter tengcongensis and transformed into Escherichia coli. The protein product showed high identity (80%) to human Ape1 nuclease, whereas to E.coli exonuclease III – 78%. This is the first prokaryotic AP nuclease that exhibits such high identity to human Ape1 nuclease. The very high expression level (57% of total soluble proteins) of fully active and soluble His₆ -tagged Tte AP enzyme with His₆ -tag on C-terminal end was obtained in Escherichia coli Rosetta (DE3) pLysS. The active enzyme was purified up to 98% homogeneity in one chromatographic step using metal-affinity chromatography on Ni²⁺-IDA-Sepharose resin. The yield was 90 mg (14 000 kU) of pure His₆ -tagged Tte AP (153 kU/mg) from 1 liter of culture. The optimal conditions of Tte AP endo-, exonuclease and 3’-nuclease activity were investigated using fluorescein labeled dsDNA with inserted AP sites and ssDNA. Optimal Tte AP endonuclease activity was observed at 70–75°C, pH 8.0 and at low Mg²⁺ concentration (0.5 mM). Higher Mg2+ concentration (> 1 mM) enhanced 3’-5’ exonuclease activity and at Mg²⁺ concentration > 2.0 mM 3’ nuclease activity was observed.Because of the endonuclease activity of Tte AP exonuclease, the enzyme was applied in PCR amplification of long DNA templates. Tte AP exonuclease eliminated AP-sites in DNA template and improved the efficiency of DNA amplification.