RAD50 promotes DNA repair by homologous recombination and restrains antigenic variation in African trypanosomes

Abstract

147 words 23 24 ABSTRACT 25 Homologous recombination dominates as the major form of DNA repair in Trypanosoma brucei, and is 26 especially important for recombination of the subtelomeric variant surface glycoprotein during 27 antigenic variation. RAD50, a component of the MRN complex (MRE11, RAD50, NBS1), is central to 28 homologous recombination through facilitating resection and governing the DNA damage response. 29The function of RAD50 in trypanosomes is untested. Here we report that RAD50 is required for 30 RAD51-dependent homologous recombination, phosphorylation of histone H2A and controlled 31 resection following a DNA double strand break (DSB). Perhaps surprisingly, DSB resection in the 32 rad50 nulls was not impaired and appeared to peak earlier than in the parental strains. Finally, we 33 show that RAD50 suppresses DNA repair using donors with short stretches of homology at a 34 subtelomeric locus, with null strains producing a greater diversity of expressed VSG variants following 35 DSB repair. We conclude that RAD50 promotes stringent homologous recombination at subtelomeric 36 loci and restrains antigenic variation. 37 38 42 vector, the tsetse fly, and mammalian hosts, where they colonise the blood, fat 1 and skin 2 and 43 eventually cross the blood brain barrier in late stage infection. If left untreated, trypanosomiasis is 44 normally fatal 3 . In the mammalian host, each trypanosome cell is covered in a dense layer of a single 45 species of variant surface glycoprotein (VSG). The highly immunogenic VSG layer 4,5 acts as an 46 barrier, concealing other surface components from the host immune response 6 . Trypanosomes 47 maintain a persistent infection by continuously escaping the host's immune response though antigenic 48 variation 7 . Central to this survival strategy is monoallelic expression of the VSG from a subtelomeric 49 locus, known as an expression site (VSG-ES), and stochastic VSG switching. The ~ 15 VSG-ESs in 50 the trypanosome genome share a high degree of sequence and structure conservation 8 , each being 51 an RNA polymerase-I (RNA Pol-I) polycistronic transcription unit with a single VSG gene found 52 adjacent to the telomere, up to 60 kb downstream of the promoter 8 . The VSG gene is flanked by two 53 sets of repetitive sequence: downstream is the telomere, and upstream is a block of repetitive 54 sequence, known as the 70-bp repeats, which modulates VSG switching 8,9 . Characteristic of a 55 trypanosome infection are recrudescent waves of parasitemia, each of which is composed of a diverse 56 VSG expressing population, with between 7 -79 VSGs detected in each peak of parasitemia 10-12 . 57 VSG diversity arises through altering the single VSG-ES that is transcribed or, more commonly, by 58 recombination of silent VSGs into the active VSG-ES. The seemingly unrestricted use of VSG genes 59 might be expected to result in a rapid exhaustion of the VSG gene repertoire. However, the parasite's 60 ability to sustain an infection appears to lie in an enormous repertoire of >2000 VSG genes ...

DNA Double-Strand Breaks: A Double-Edged Sword for Trypanosomatids

“…First, the recombination-based VSG switching can be directly activated by the induction of a DSB in the active BES using the meganuclease I-SceI (Boothroyd et al, 2009;Glover et al, 2013). Second, disruption of the HR pathway through the interruption of some components, such as ATR (Stortz et al, 2017;Black et al, 2020;Marin et al, 2020), Rad51 (McCulloch andProudfoot and McCulloch, 2005), Rad50 (Mehnert et al, 2021), and BRCA2 (Hartley and McCulloch, 2008), impairs the VSG switching by recombination, suggesting that multiple components are shared between these two pathways. In general, these features mirror targeted gene rearrangements in other organisms, such as VAR genes diversity in Plasmodium (Kyes et al, 2007;Claessens et al, 2014), pilin antigenic variation in Neisseria (Cahoon and Seifert, 2011), and V(D)J recombination during the development of B lymphocytes of the vertebrate immune system (Tonegawa, 1983;Brecht et al, 2020).…”

“…Trypanosomatids (supergroup Excavata) have most of their DNA repair pathways conserved. However, notable divergencies exist suggesting a parasite-specific repurposing of the DSBs repair machinery (Glover et al, 2013;Ubeda et al, 2014;Alves et al, 2018;Mehnert et al, 2021). While the HR repair pathway is conserved and functional (McCulloch and Glover et al, 2008;Hartley and McCulloch, 2008;Genois et al, 2012;Alves et al, 2018;Marin et al, 2018), canonical NHEJ activities appear absent in trypanosomatids (Burton et al, 2007;Nenarokova et al, 2019).…”