Date of Award

2010-01-01

Degree Name

Master of Science

Department

Biological Sciences

Advisor(s)

Renato Aguilera

Abstract

Nucleases are enzymes that breakdown nucleic acids; they are classified by their biochemical properties into different groups (Evans et al., 2003). The ββα-Me finger family of nucleases are enzymes that combine structurally different groups but they are defined by a highly evolutionary conserved active site, a stretch of 22 amino acids composed by a β-hairpin (ββ) and α-helix (α) that anchor a catalytic metal ion (Sokolowska et al, 2009). Within this family is found the DNA/RNA non-specific nucleases. Nucleases that belong to the DNA/RNA non-specific group hydrolyze both ds and ss DNA, as well as RNA at equal or similar rates (Rangarajan and Shankar, 2001). They all share a common structure, specifically, a DNA/RNA non-specific endonuclease (NUC) domain containing a conserved Asp-Arg-Gly-His (DRGH) motif with an active site histidine (Friedhoff et al., 1994, 1996, Meiss et al., 2000).

In a recent phylogenetic study on Kamchatka crab duplex-specific nuclease (Par_DSN), a new classification of Par_DSN-like nucleases has been proposed. Conserved regions of the NUC domains from the alignment data were used for ML tree construction with TREE-PUZZLE software (Anisimova et al., 2008). The NUC domains examined were divided into two main subgroups, the first containing SFN members and two insect enzymes, and the second (that they called duplex-specific nucleases, DS_NUCs) consisting of other insect and Crustacean nucleases (Anisimova et al., 2008). Among this last group there are two nucleases that were uncover by our group in Drosophila melanogaster via microarray analysis annotated as: CG33346 and CG9989.

Deoxyribonuclease II (DNase II) is an endonuclease with optimal activity at low pH. It is localized within the lysosomes of higher eukaryotes for degradation of DNA after phagocytosis. We have previously demonstrated that down regulation of the Drosophila melanogaster dnase II gene, using RNA interference (RNAi) resulted in increased susceptibility to bacterial infection. In addition, genome wide expression microarray analyses of infected DNase II-deficient flies revealed over expression of a DNase-like gene that has been tentatively named dnase IIIa. Interestingly, a second highly homologous gene, that we have named dnase IIIb, was found adjacent to dnase IIIa.

This evidence suggests that dnase IIIa could be up-regulated to compensate for the lost of DNase II and function as antimicrobial response proteins. In this study we will focus on the dnase IIIb gene since this ORF seems to encode an authentic nuclease that can be up-regulated during bacterial infection. The putative dnase IIIb gene was cloned and modified to contain a GST and nine-Histidine-tag and inserted into the bacterial-expression vector pGEX-KG. DNase IIIβ was induced by addition of IPTG and the recombinant protein was then purified by GST affinity column and found to be enzymatically active. By characterizing this novel nuclease, we will be able to further determine the biological function of this enzyme during Gram-positive bacterial infection.

Language

en

Provenance

Received from ProQuest

File Size

63 pages

File Format

application/pdf

Rights Holder

Brenda Cristina Anchondo

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