Aparna Sunder and Erik D.

Larson School of Biological Sciences, Illinois State University, Normal IL 61790-4120

CHARACTERIZATION OF HUMAN ENDONUCLEASE V

INTRODUCTION, BACKGROUND AND SIGNIFICANCE

ABSTRACT Endonuclease V (ENDO V) is a highly conserved DNA repair protein, yet its roles in maintaining genomic stability are not defined. Mutational analysis in E. coli suggests ENDO V is involved in the repair of nitrosative DNA damage1, 2, but it has also been proposed that ENDO V may function in tandem with the base excision repair pathway to correct a wide range of DNA base modifications3. Biochemically, it has been demonstrated that Thermotoga maritima ENDO V nicks duplex DNA containing deoxyinosine, abasic sites, base mismatches, or uracil at the second phosphodiester bond 3 to a deaminated base6, supporting roles in correcting DNA base modifications. In a vertebrate model, murine ENDO V may have a role in the removal of Hypoxanthine from DNA4, however the function of ENDO V in eukaryotes has not been defined. I have identified a hypothetical protein FLJ35220 (GenBank ID BC064545) with a predicted nucleotide sequence of 795 bp and an amino acid sequence of 264 aa5 , which shows significant homology to the ENDO V of E.coli, Pyrococcus furiosus, Thermotoga maritima, Mus musculus and putative ENDO V of Gallus gallus. I have cloned the gene for the human homolog of ENDO V for expression in E. coli. Purified recombinant human ENDO V (HsENDOV) incubated with a synthetic oligonucleotide with deoxyinosine suggests that HsENDOV may have a role in repairing deoxyinosine in single stranded DNA substrates in humans. In order to confirm cleavage activities are due to ENDO V, I have constructed a HsENDOV D52A point mutation using site directed mutagenesis and introduced it into the same E. coli expression system as the wild type (wt). Cleavage assays comparing wt and mutant HsENDOV will test the model that the FLJ35220 expression clone represents the human homolog of ENDO V. HYPOTHESIS ENDO V is a highly conserved DNA repair protein and HsENDOV functions to trigger correction of DNA damage induced by reactive nitrogen species. To test this hypothesis, I will take the following approaches Aim 1. Characterization HsENDOV activity of METHODS Making an expression clone of human ENDOV in the E.coli expression host BL21 (STAR) DE3 for IPTG inducible protein expression. Sequencing the clone Purification of active recombinant human ENDOV. Designing the substrate for cleavage assays Cleavage assays and resolution of cleavage products by denaturing PAGE and imaging by phosphorimager (GE, Amersham). Figure 2. Proposed model for the action of Endonuclease V 1. dsDNA containing deoxyinosine (deaminated Adenine) 2. ENDO V will nick the damaged strand at the second phosphodiester bond 3 from the lesion 3. A 3-5 Exonuclease will cleave the damaged strand and remove the lesion to produce gaps 4. DNA polymerase and ligase will fill the gaps, resynthesize the nucleotides and complete the repair Aspartate 52 to Alanine mutation of human ENDOV using Phusion site directed mutagenesis kit from New England Biolabs. Making an expression clone of the mutant form in the E.coli expression host BL21 (STAR) DE3. Expression and purification of mutant recombinant human ENDOV. Comparative cleavage assays with wild type and mutant recombinant human ENDOV to establish the impact of the mutation on the catalytic residue.

Aim 2. Defining the catalytic residue of HsENDOV Upon completion of these goals, I will have identified the human homolog of Endo V and determined its function. Currently it is not known how Endo V facilitates repair in any organism. In the Archaeal organism, Thermotoga maritima, the crystal structure has been solved9. Figure 1. Crystal structure of Thermotoga maritima Endonuclease V Green tube DNA backbone Pale yellow ball and stick nucleotide side chains Orange spheres protein backbone Blue wires amino acid side chains Magenta sphere magnesium ion Highlighted yellow spheres amino acid residues involved in DNA recognition (His214), metal binding (Asp43), phosphodiester incision (Glu89, Asp110), and hypoxanthine lesion recognition pocket(Leu85, Gly111, Gln112, Gly113, Gly136 and Leu142)

RESULTS
Figure 5(A). HsENDOV cleavage of single stranded oligonucleotide substrate containing deoxyinosine Recombinant HsENDOV and PfuENDOV, both show a 26-mer cleavage product, which is in agreement with the size of EcENDOV cleavage product run alongside as a positive control. However, there is also a 1nt cleavage product, which was verified to be due to a 3-5 exonuclease activity by comparing with the known activity of Mung bean exonuclease (data not shown here).

CONCLUSION AND REFERENCES

CONCLUSIONS Human hypothetical protein FLJ35220 could indeed be Endonuclease V based on sequence homology with species representative of all kingdoms. Recombinant HsENDOV can be expressed in an E.coli host system. Recombinant PfuENDOV can be expressed in an E.coli host system. Active purified recombinant human ENDOV and Pfu ENDOV have been obtained (activity tested on a single stranded substrate containing deoxyinosine). A 3-5 exonuclease activity has been observed in the purified recombinant proteins, which could be an activity of ENDO V itself. However this model needs more conclusive experimental proof. In order to prove, cleavage of the damaged substrate is indeed due to HsENDOV, an Aspartate to Alanine subsitution mutation has been engineered by site directed mutagenesis. Loss of activity due to the mutation is yet to be established. FUTURE GOALS Genetic complementation studies on E.coli nfi mutants Studies on cellular localization of HsENDOV in the human Byrkitt lymphoma cell line Ramos

REFERENCES 1. E.C. Friedberg et al., DNA Repair and Mutagenesis. 2, 379, 381 383 (2006). 2. G.Guo, B.Weiss, J. Bacteriol. 180, 46 (1998). 3. B.Weiss, Mutation Research. 461, 301 (2001). 4. H.Feng et al., Biochemistry. 45, 10251 (2006). 5. H.Feng et al., Biochemistry. 44, 11486 (2005). 6. Kanugula et al., Proc. Natl. Acad. Sci. U.S.A. 102, 3617 (2005). 7.http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=4055576 6 Accessed on 1/26/09. 8. F.T.Gates, III., S.Linn, J. Biol. Chem. 252, 2802 (1977). 9. B.Dalhus et al., Nature Structural & Molecular Biology 16, 138 - 143 (2009).

Figure 3. ENDOV - Conserved amino acid residues in representative organisms from various kingdoms From the NCBI database, I have identified a hypothetical human protein FLJ35220 (GenBank ID BC064545) with a predicted nucleotide sequence of 795 bp and an amino acid sequence of 264aa7 , which shows significant homology to the ENDO V of E.coli (EcENDOV), putative Endo V of Pyrococcus furiosus (PfuENDOV), putative Endo V of Gallus gallus (GgENDOV) and Mus musculus (MmENDOV). Conserved amino acid residues with 100% identity are highlighted in red.

Figure 4. Purification of recombinant HsENDOV and PfuENDOV on Nickel Sepharose resin Top purification of recombinant HsENDOV wild type in E.coli host. Middle purification of recombinant PfuENDOV in E.coli host. Bottom panel purification of the D52A mutant recombinant HsENDOV in E.coli host.

Figure 5(B). HsENDOV cleavage of single stranded oligonucleotide substrate containing deoxyinosine in the presence of a competitive inhibitor Another substrate EDL409, identical to EDL410, but an unlabeled 51-mer oligonucleotide with Adenine in the place of deoxyinosine in other words, an undamaged substrate was used in the experiment as a competitive inhibitor in decreasing concentrations from 1000 fold excess down to equimolar proportions to titrate off the exonuclease activity and get only the proposed Endonuclease V activity.