de novo creation of base J in Leishmania

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ABSTRACT There are over 20 pathogenic species of the genus Leishmania that are the causative agent for leishmaniasis. Symptoms for leishmaniasis range from self-limiting cutaneous lesion to visceral leishmaniasis that is fatal if left untreated. Globally there are more than 1 million reported cases in the last 5 years with over 20,000 fatalities reported annually. Leishmania resides in one of the most deeply branched eukaryotic lineages and harbors a number of distinct and novel biological processes, two of which are important for this proposal. The first is the organization of the protein coding genes transcribed by RNA polymerase II into long clusters of unrelated genes. The cluster of genes are transcribed as a single primary RNA from a transcription start site. Transcription then proceeds through the polycistronic cluster of genes with the individual mRNAs processed by trans-splicing until it reaches a transcription termination site. The second process is the modification of ~1% of thymidines (T) in DNA with glucose to form β-D-glucosyl-hydroxymethyluracil (J). The modification of T to J is a two-step process with the hydroxylation of T by either JBP1 or JBP2. Glucose is subsequently added to the hydroxyl group by a specific glucosyltransferase. JBP1 and JBP2 have distinct roles in the formation of J. JBP1 is responsible for maintenance of J, in regions already containing J, following DNA synthesis on the newly formed unmodified strand. JBP2 is responsible for de novo insertion of J, mostly at sites missed by JBP1, and ultimately determines which bases are modified to J. Most of J (~99%) is localized to the telomeres, but J is also found at internal sites most notably at all but one of the transcription termination sites. The modification of T to J is essential in Leishmania, so a deeper understanding of how Leishmania determines which bases are modified will be crucial in the development of potential therapeutic targeting the creation of J. The protein sequence of JBP2 does not contain any motif that would allow it to bind DNA, but it does contain a protein interaction domain. We determined that JBP2 interacts in a complex with a protein we named J2TDP. The protein sequence of J2TDP contains a Tudor domain, a motif known to interact with protein that contain dimethylated lysines or arginines, which are principally found in histones. The identification of J2TDP led us to the hypothesize that J2TDP binds a specific histone modification and then recruits JBP2 to the chromosome to modify adjacent Ts for conversion into J. In this proposal we break this hypothesis down in the follow three sub-hypotheses and then test key predictions of the hypotheses. 1) J2TDP binds a histone modification. 2) J2TDP recruits JBP2 to the chromosome. 3) Localization of JBP2 to the chromosome is sufficient for localized modification to J.