Partial purification and characterization of DNA polymerase beta-like enzyme from Plasmodium falciparum.
DNA polymerases play crucial roles, not only in DNA replication, transcription and recombination, but also in DNA repair to maintain the integrity of the cell's genome. In Plasmodium falciparum, only three types of DNA polymerases-alpha, gamma, and delta have previously been characterized, whereas DNA polymerase beta, the major enzyme operating during base excision repair in eukaryotes, has yet to be isolated and characterized. In this study, DNA polymerase beta-like activity was detected in crude extract of P. falciparum trophozoites. P. falciparum DNA polymerase beta-like enzyme was partially purified using fast protein liquid chromatography, with a yield of 2.8% and 825-fold purification. The partially purified enzyme was highly resistant to aphidicolin and N-ethylmaleimide, as in other eukaryotic enzymes, but was also resistant to 2',3'-dideoxythymidine-5'-triphosphate and to other synthetic nucleoside analogs. The parasite enzyme showed low processivity. Using UG mismatch substrate to investigate base excision repair, the P. falciparum DNA polymerase beta-like enzyme could repair a patch size of 3-5 nucleotides, indicative of involvement in a long patch repair pathway, the first evidence of such a property in the DNA polymerase of a malaria parasite.
Nunthawarasilp
Prapa
P
Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand.
Petmitr
Songsak
S
Chavalitshewinkoon-Petmitr
Porntip
P
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D002853:Chromatography, Liquid; D019951:DNA Polymerase beta / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004260:DNA Repair; D016054:DNA, Protozoan / Q000235:genetics; D054306:Dideoxynucleotides; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D010963:Plasmodium falciparum / Q000201:enzymology*; D015800:Protozoan Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D013942:Thymine Nucleotides / Q000494:pharmacology
D016054:DNA, Protozoan; D054306:Dideoxynucleotides; D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D013942:Thymine Nucleotides; D016590:Aphidicolin; D019951:DNA Polymerase beta; C017216:2',3'-dideoxythymidine triphosphate; D005033:Ethylmaleimide