NEIL Gene Knockouts: Correlation with mutation frequency. Illustrate your essay with specific examples.

Review of literature

Endogenous and exogenous ROS (reactive oxygen species) are responsible for causing damage to different cellular components especially DNA at the rate of 104 molecular lesions per cell per day. A number of disease etiology mechanisms are tied to mutations in mitochondrial or nuclear DNA (Bandaru et al, 2002). The Base Excision Repair mechanism is the first line of defense and consists of enzymes like NEIL1, NEIL2 and NEIL3 (nei endonuclease VIII-like 1, 2 and 3 respectively) that facilitate DNA damage repair (Conlon et al, 2005; Hazra et al, 2002; Morland et al, 2002). Studies have shown that deletion or mutation of genes coding these enzymes leads to a greater incidence of tumours. These enzymes function as DNA glycosylase and apyrimidinic/apurinic lyase catalyse beta and delta-elimination reaction steps in the DNA around the damaged base, generate 3′ terminal phosphate and interact with DNA ligase III alpha, BER and DNMA Polymerase beta. Additional function include repair of bubble DNA/single-strand DNA for oxidised bases, stimulate production of another vital repair enzyme OGG1 by carrying our beta/delta elimination similar to APE1 at the AP site and excision of excises 5-hydroxyuracil and 3′ end proximal 8-hydroxyguanine lesions.

Aims and Objectives

The overall aim is to generate in vitro and in vivo models for the three NEIL genes and observe the phenotype and correlate this with propensity to mutagenicity, especially expanded simple tandem repeat (ESTR) germ line mutations using cytotoxicity and apoptosis assays. The plan also included generation of NEIL2 knockouts and investigation on the possibility of NEIL3 knockouts.

Design and Methodology

We have successfully generated NEIL1 knockouts in the lab. We are currently in the process of generating NEIL 2 knockouts. We plan to test efficacy of knockout using enzymatic assays. There are however, problems other than tumour incidence in NEIL gene knockouts. It has been shown that NEIL1 knockout mice develop metabolic syndrome characterised by hyperinsulinemia, obesity and dyslipidemia (Shinmura et al, 2004; Vartanian et al, 2006). In addition generating NEIL2 knockout is difficult. We are planning to address these issues by using conditional knockout using the Tet on or Tet off system or by RNA interference. This will enable us to selectively switch on and switch off the NEIL genes upon requirement for a shorter duration rather than having a permanent knockout. We are also interested in the NEIL3 gene function since it is known to have no DNA glycosylase activity and is implicated in HIV infection (Hildrestrand et al, 2009). Also it is expressed only in immunological cells. But it is not known as to what protein partners it interacts. We plan to address this using protein pull downs and gene protein interaction methods.

Sources and data acquisition

Data for the present proposal was collected from PUBMED, Medline, Biomednet, and other medical databases.