Involvement of a type 2 ribonuclease H in kinetoplast maxicircle DNA replication in Trypanosoma brucei (LB165)

Abstract

Ribonuclease H (RNase H, EC # 3.1.26.4) catalyzes the hydrolysis of RNA from a DNA/RNA hybrid. This ubiquitous enzyme interacts with several different DNA‐modifying enzymes in the cell and participates in the replication, recombination and repair of nuclear and mitochondrial DNA molecules. We report here that the type 2 RNase H of the parasitic protozoan Trypanosoma brucei is a predominantly mitochondrial enzyme and is essential for the survival of both the procyclic and the blood stream forms. Yeast 2‐hybrid and co‐IP analyses for the mitochondrial DNA replication‐associated proteins interacting directly with RNase HIIC revealed binding of RNase HIIC with the mitochondrial DNA ligase Kα which is involved in kinetoplast DNA replication. Interestingly, we found that RNase HIIC also binds with the DNA recombinase DMC1 which is also located in the mitochondrion of the parasite. Chromatin immunoprecipitation data indicated binding of both RNase HIIC and DMC1 to the maxicircle DNA. We found that the maxicircle DNA and transcripts derived from it are decreased significantly in the cells with decreased levels of RNase HIIC. This enzyme is required for the recovery from DNA damage caused by caffeine, hydroxyurea and methylmethane sulfonate, as the RNase HIIC knocked down cells are more sensitive to the lethality of these DNA damaging agents. We conclude that RNase HIIC collaborates with DNA ligase Kα and DMC1 to participate in the replication and repair of mitochondrial maxicircle DNA in these parasite cells. This research was supported in parts by NIAID grants 5R01AI042327, 1R21AI076757‐01A1 and NIGMS grant #3 S06 GM008037‐34‐S2 to GC.