Abstract
DNA damage and repair in trypanosomatids impacts virulence, drug resistance and antigenic variation but, currently, little is known about DNA damage responses or cell cycle checkpoints in these divergent protozoa. One of the earliest markers of DNA damage in eukaryotes is γH2A(X), a serine phosphorylated histone H2A (variant). Here, we report the identification and initial characterization of γH2A in Trypanosoma brucei. We identified Thr130 within the replication-dependent histone H2A as a candidate phosphorylation site and found that the abundance of this trypanosomal γH2A increased in vivo in response to DNA damage. Nuclear γH2A foci mark the sites of putative natural replication fork stalling, sites of meganuclease-induced DNA double strand breaks and sites of methyl methanesulphonate-induced DNA damage. Naturally occurring and meganuclease-induced γH2A and RAD51 double-positive repair foci are typically found in S-phase or G2 nuclei. The results link trypanosomal γH2A, with an unusual histone modification motif, to DNA damage sensing and mitotic checkpoint signaling.
Highlights
DNA rearrangement in trypanosomatids can bring about changes in virulence and drug resistance and is well known for its role in switching variant surface glycoprotein expression and antigenic variation in Trypanosoma brucei [1]
RAD51, with Green Fluorescent Protein fused to the C-terminus, was expressed using the pNATRAD51-GFP construct, a derivative of pNATxGFP [22]. pNATRAD51-GFP was linearized by digestion with NsiI prior to transfection
Our findings indicate that ␥H2A foci are formed in response to natural DNA lesions, meganuclease-mediated DNA double strand breaks, and chemical DNA damage
Summary
DNA rearrangement in trypanosomatids can bring about changes in virulence and drug resistance and is well known for its role in switching variant surface glycoprotein expression and antigenic variation in Trypanosoma brucei [1]. A number of T. brucei factors have demonstrated roles in homologous recombination [1], and site-specific cleavage, following inducible expression of the yeast I-SceI meganuclease, has proven to be a powerful tool, allowing repair monitoring [2], investigation of mechanisms of antigenic variation [4], and genetic dissection of repair pathways [3]. Studies in this area are restricted, due to the relative paucity of reagents available to investigate DNA damage and repair processes in trypanosomatids
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
