Developing a Catalytically Functional Ribonucleotide Reductase for Neurospora Crassa that Lacks Heterokaryon Incompatibility Activity

We are exploring the structure-function relationship of proteins governing nonself recognition in Neurospora crassa. The heterokaryon incompatibility locus het-6, from N. crassa comprises two tightly linked genes: un-24 and het-6. Each gene exists as either Oak Ridge (OR) or Panama (PA) allelic variants, and only two possible haplotypes occur in nature: un-24OR-het-6OR or un-24PA-het-6PA. The het-6 gene encodes a protein with no known function outside of incompatibility whereas un-24 also encodes the large subunit of ribonucleotide reductase (RNR). RNR is an enzyme that is essential for the conversion of ribonucleotides to deoxyribonucleotides, and is crucial for DNA synthesis. We identify a single glutamic acid residue within the C-terminus of UN-24OR that governs incompatibility specificity. By changing this glutamic acid to leucine we produced an enzymatically functional RNR that lacks incompatibility activity (UN-240). Using this un-240 strain, we provide novel insight into the role that HET-6 proteins play in nonself recognition.