Discussion

During transcription, nascent mRNA can bind to the template strand of unwound DNA, forming thermodynamically stable R loops. Although threatening to stability, R loops are powerful regulators of DNA replication, gene expression and recombination. In humans, the most efficient origins of replication (ORIs) localise to CpG island promotors. These sites form R loops upon transcription and bind the origin recognition complex, ORC. T. brucei co-transcribes its nuclear genes from multigene clusters, with adjacent clusters separated by poorly characterised promoters and terminators at so called strand switch regions (SSRs). Transcription and replication appear to be linked as ORIs localise to select SSRs and ORC binds to potentially all SSRs. Given this, we are investigating whether R loops in T. brucei sequester ORC and direct DNA replication. In order to preserve R loops, we have generated mutants deficient in two nuclear RNase H genes that digest hybridised RNA and will describe the impact on growth and DNA synthesis. We are also employing ChIP-seq technology map R loops across the genome. In addition, R loops facilitate class switching in activated B lymphocytes to generate antibody diversity, a system which may be analogous to variant surface glycoprotein switching in T. brucei. Hence, we will describe the effect of RNase H mutation on antigenic variation. Finally, we will describe a third apparently catalytic RNase H, localised to the antipodal sites of the kinetoplast DNA.