Quantitative solid-phase assay to measure deoxynucleoside triphosphate pools.

deoxynucleoside triphosphate (dNTPs) are the reduced nucleotides used as the building blocks and energy source for deoxyribonucleic acid (DNA) replication and maintenance in all living systems. They are present in highly regulated amounts and ratios in the cell, and their balance has been implicated in the most important cell processes, from determining the fidelity of DNA replication to affecting cell fate. Furthermore, many cancer drugs target biosynthetic enzymes in dNTP metabolism, and mutations in genes directly or indirectly affecting these pathways that are the cause of devastating diseases. The accurate and systematic measurement of these pools is key to understanding the mechanisms behind these diseases and their treatment. We present a new method for measuring dNTP pools from biological samples, utilizing the current state-of-the-art polymerase method, modified to a solid-phase setting and optimized for larger scale measurements.

A complex genomic locus drives mtDNA replicase POLG expression to its disease-related nervous system regions.

DNA polymerase gamma (POLG), the mtDNA replicase, is a common cause of mitochondrial neurodegeneration. Why POLG defects especially cause central nervous system (CNS) diseases is unknown. We discovered a complex genomic regulatory locus for POLG, containing three functional CNS-specific enhancers that drive expression specifically in oculomotor complex and sensory interneurons of the spinal cord, completely overlapping with the regions showing neuronal death in POLG patients. The regulatory locus also expresses two functional RNAs, LINC00925-RNA and MIR9-3, which are coexpressed with POLG The MIR9-3 targets include NR2E1, a transcription factor maintaining neural stem cells in undifferentiated state, and MTHFD2, the regulatory enzyme of mitochondrial folate cycle, linking POLG expression to stem cell differentiation and folate metabolism. Our evidence suggests that distant genomic non-coding regions contribute to regulation of genes encoding mitochondrial proteins. Such genomic arrangement of POLG locus, driving expression to CNS regions affected in POLG patients, presents a potential mechanism for CNS-specific manifestations in POLG disease.