Initiation of DNA replication in Trypanosoma brucei is regulated by the interaction of the RNaseH2 complex with DOT1 histone methyltransferases

Abstract text

Post-translational histone modifications such as lysine methylation influence the structure and the function of chromatin. DOT1 (disruptor of telomeric silencing 1) is a highly-conserved methyltransferase that exclusively methylates histone H3 lysine K79 (H3K79). T. brucei has two homologues, DOT1A and DOT1B, with differences in product specificity and function. While DOT1B mediates mono-, di-, and trimethylation of H3K76, DOT1A only catalyses mono- and di-methylation of this residue (H3K76me1/2). We showed previously that H3K76me1/2 is restricted to G2 phase and mitosis, and that DOT1A is a master regulator of replication initiation. RNAi-mediated depletion of DOT1A abolishes DNA replication completely whereas overexpression of DOT1A causes re-initiation of replication before the cell has completed S phase, implicating DOT1A as a master regulator of replication initiation. Since conserved regulatory domains are absent in these enzymes in T. brucei, the mechanism of cell cycle-dependent activity of DOT1 enzymes has remained elusive. Recently, we discovered that the ribonuclease H2 complex (RNaseH2) interacts with DOT1 enzymes in T. brucei. In vitro studies with recombinant enzymes and reconstituted nucleosomes demonstrated that RNaseH2 is an inhibitor of DOT1A activity. Using x-ray footprinting, we identified the interface region of the DOT1A/RNaseH2 complex and were able to abolish the inhibitory effect of RNaseH2 by introducing site-specific mutations into this site. Further experiments in vivo suggested that RNaseH2 interactions with DOT1 enzymes orchestrates their cell cycle-dependent activity in T. brucei, representing a novel mechanism of replication regulation.