Reduced methionine synthase expression results in uracil accumulation in mitochondrial DNA and impaired oxidative capacity

Katarina E Heyden; Joanna L Fiddler; Yuwen Xiu; Olga V Malysheva; Michal K Handzlik; Whitney N Phinney; Linsey Stiles; Sally P Stabler; Christian M Metallo; Marie A Caudill; Martha S Field

doi:10.1093/pnasnexus/pgad105

Reduced methionine synthase expression results in uracil accumulation in mitochondrial DNA and impaired oxidative capacity

PNAS Nexus. 2023 Mar 27;2(4):pgad105. doi: 10.1093/pnasnexus/pgad105. eCollection 2023 Apr.

Authors

Affiliations

¹ Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853, USA.
² Department of Bioengineering, University of California, La Jolla, San Diego, CA 92093, USA.
³ Department of Medicine, University of Colorado, Aurora, CO 80217, USA.
⁴ Department of Medicine/Division of Endocrinology, University of California, Los Angeles, CA 90095, USA.
⁵ Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037, USA.

Abstract

Adequate thymidylate [deoxythymidine monophosphate (dTMP) or the "T" base in DNA] levels are essential for stability of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). Folate and vitamin B12 (B12) are essential cofactors in folate-mediated one-carbon metabolism (FOCM), a metabolic network which supports synthesis of nucleotides (including dTMP) and methionine. Perturbations in FOCM impair dTMP synthesis, causing misincorporation of uracil (or a "U" base) into DNA. During B12 deficiency, cellular folate accumulates as 5-methyltetrahdryfolate (5-methyl-THF), limiting nucleotide synthesis. The purpose of this study was to determine how reduced levels of the B12-dpendent enzyme methionine synthase (MTR) and dietary folate interact to affect mtDNA integrity and mitochondrial function in mouse liver. Folate accumulation, uracil levels, mtDNA content, and oxidative phosphorylation capacity were measured in male Mtr^+/+ and Mtr^+/- mice weaned onto either a folate-sufficient control (C) diet (2 mg/kg folic acid) or a folate-deficient (FD) diet (lacking folic acid) for 7 weeks. Mtr heterozygosity led to increased liver 5-methyl-THF levels. Mtr^+/- mice consuming the C diet also exhibited a 40-fold increase in uracil in liver mtDNA. Mtr^+/- mice consuming the FD diet exhibited less uracil accumulation in liver mtDNA as compared to Mtr^+/+ mice consuming the FD diet. Furthermore, Mtr^+/- mice exhibited 25% lower liver mtDNA content and a 20% lower maximal oxygen consumption rates. Impairments in mitochondrial FOCM are known to lead to increased uracil in mtDNA. This study demonstrates that impaired cytosolic dTMP synthesis, induced by decreased Mtr expression, also leads to increased uracil in mtDNA.

Keywords: DNA; folate; methionine synthase; uracil; vitamin B12.