Biochemistry

Abstract

The DNA polymerase beta (DNA pol b) plays a central role in base excision repair and could also interfere in other aspects of DNA repair and metabolism when overexpressed in mammalian cells. Nothing is known about the role of this enzyme and its molecular significance for the Trypanosoma cruzi biology. We have cloned and sequenced the DNA pol b from this organism. The TcDNA pol b sequence has 66% homology with the Chritidia fasciculata and Leishmania (L.) infantum DNA pol b genes. The TcDNA pol b is able to clomplement the Escherichia coli clone SC18-12 deficient in the polA -gene. The expression of TcDNA pol b in those bacterial cells restores their ability to grow at 42ºC and diminishes their sensibility to U.V. radiation. In addition, the overexpression of TcDNA pol b in CHO cells provokes an increase in the cells mutation rate in the 6-TG resitence assay. The TcDNA pol b protein has been expressed in E. coli in fusion with MBP and purified in a maltose column. The TcDNA pol b fusion protein was capable to add the dNTPs to primer in a DNA extension assay. Futhermore, this enzyme was used an assay of 3 H-dTTP incorporation measured by CPM counting to characterize its behavior in relation to dNTPs concentration. The enzyme works in optimal concentration of 5mM of dNTPs. This same assay has been used to demostrate that this enzyme has the ability to incorporate the modified nucleotide AZTTP to DNA molecules. So, we have cloned and characterized the DNA pol b gene from Trypanosoma cruzi and have started biochemical characterization of this gene product.