Disease-Associated Mutations G589A and V590F Relieve Replication Focus Targeting Sequence-Mediated Autoinhibition of DNA Methyltransferase 1
In eukaryotes, the most common epigenetic DNA modification is methylation of carbon 5 of cytosines, predominantly in CpG dinucleotides. Methylation patterns are established and maintained by a family of proteins known as DNA methyltransferases (DNMTs). DNA methylation is an important epigenetic mark associated with gene repression, and disruption of the normal DNA methylation pattern is known to play a role in several disease states. Methylation patterns are primarily maintained by DNMT1, which possesses specificity for methylation of hemimethylated DNA. DNMT1 is a multidomain protein with a C-terminal catalytic methyltransferase domain and a large N-terminal regulatory region. The replication focus targeting sequence (RFTS) domain, found in the regulatory region, is an endogenous inhibitor of DNMT1 activity. Recently, several mutations in the RFTS domain were shown to be causal for two adult onset neurodegenerative diseases; however, little is known about the impact of these mutations on the structure and function of DNMT1. Two of these mutations, G589A and V590F, are associated with development of autosomal dominant cerebellar ataxia, deafness, and narcolepsy (ADCA-DN). We have successfully expressed and purified G589A and V590F DNMT1 for in vitro studies. The mutations significantly decrease the thermal stability of DNMT1, yet the mutant proteins exhibit 2.5–3.5-fold increases in DNA binding affinity. In addition, the mutations weaken RFTS-mediated inhibition of DNA methylation activity. Taken together, these data suggest these disease-associated mutations decrease protein stability and, at least partially, relieve normal RFTS-mediated autoinhibition of DNMT1.
Dolen, Emma K.; McGinnis, James H.; Tavory, Rachel N.; Weiss, Jill; and Switzer, Rebecca L.. "Disease-Associated Mutations G589A and V590F Relieve Replication Focus Targeting Sequence-Mediated Autoinhibition of DNA Methyltransferase 1." (2019) : 5151-5159.