Genome-Wide Stochastic Adaptive DNA Amplification at Direct and Inverted DNA Repeats in the Parasite Leishmania Jean-Michel Ubeda ¤a. , Fre ´de ´ ric Raymond . , Angana Mukherjee ¤b. , Marie Plourde, He ´le ` ne Gingras, Gae ´ tan Roy, Andre ´ anne Lapointe, Philippe Leprohon, Barbara Papadopoulou, Jacques Corbeil, Marc Ouellette* Centre de Recherche en Infectiologie, Centre de Recherche du CHU de Que ´bec, Que ´bec, Canada Abstract Gene amplification of specific loci has been described in all kingdoms of life. In the protozoan parasite Leishmania, the product of amplification is usually part of extrachromosomal circular or linear amplicons that are formed at the level of direct or inverted repeated sequences. A bioinformatics screen revealed that repeated sequences are widely distributed in the Leishmania genome and the repeats are chromosome-specific, conserved among species, and generally present in low copy number. Using sensitive PCR assays, we provide evidence that the Leishmania genome is continuously being rearranged at the level of these repeated sequences, which serve as a functional platform for constitutive and stochastic amplification (and deletion) of genomic segments in the population. This process is adaptive as the copy number of advantageous extrachromosomal circular or linear elements increases upon selective pressure and is reversible when selection is removed. We also provide mechanistic insights on the formation of circular and linear amplicons through RAD51 recombinase-dependent and -independent mechanisms, respectively. The whole genome of Leishmania is thus stochastically rearranged at the level of repeated sequences, and the selection of parasite subpopulations with changes in the copy number of specific loci is used as a strategy to respond to a changing environment. Citation: Ubeda J-M, Raymond F, Mukherjee A, Plourde M, Gingras H, et al. (2014) Genome-Wide Stochastic Adaptive DNA Amplification at Direct and Inverted DNA Repeats in the Parasite Leishmania. PLoS Biol 12(5): e1001868. doi:10.1371/journal.pbio.1001868 Academic Editor: Philip J. Hastings, Baylor College of Medicine, United States of America Received December 17, 2013; Accepted April 11, 2014; Published May 20, 2014 Copyright: ß 2014 Ubeda et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded in part by the CIHR group grant to MO, BP, and JC and by CIHR operating grants 13233 and 15501 to MO. JMU and AM were Training Fellows of the Strategic Training Program in Microbial Resistance, a partnership of the CIHR Institute of Infection and Immunity and the Fonds de Recherche en Sante ´ du Que ´bec. FR was the recipient of a CIHR studentship. JC holds the Canada Research Chair in Medical Genomics. MO is a Burroughs Wellcome Fund Scholar in Molecular Parasitology and holds the Canada Research Chair in Antimicrobial Resistance. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: CNV, copy number variation; DRs, direct repeated sequences; FBS, fetal bovine serum; HR, homologous recombination; IRs, inverted repeats; MTX, methotrexate; RAG, repeat alignment group; SIDERs, short interspersed degenerate retroposons. * E-mail: Marc.Ouellette@crchul.ulaval.ca . These authors contributed equally to this work. ¤a Current address: Centre de Ge ´ne ´tique et de Physiologie Mole ´culaire et Cellulaire UMR5534, Universite ´ Claude Bernard Lyon 1, Villeurbanne, France ¤b Current address: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America Introduction Copy number variations (CNVs) account for a substantial amount of genomic variability in mammalian genomes (reviewed in [1]). DNA amplification, a contributor of CNVs, has been reported in response to various stresses or after altered growth conditions, and can lead to extensive and often reversible genetic variations (reviewed in [2,3]). Several models have been proposed to explain DNA amplification mechanisms [2–4]. Extrachromo- somal circular DNAs can be the products of gene amplification in mammalian cells and in the protozoan parasite Leishmania (reviewed in [5,6]). In Leishmania, DNA circles are generated by homologous recombination (HR) between direct repeated se- quences (DRs) (Figure 1A) [7,8]. DNA amplification can also lead to palindrome formation. Increasing evidence suggests that palindromes are initiated at the level of inverted repeats (IRs). Indeed IRs are known to increase chromosome instability during replication, leading to hairpin formation and representing a substantial source of DNA breakage and rearrangement (Figure 1B). IRs have been shown to initiate inverted duplications in yeast cells [9–11], in protozoa [4,7,8], and in mammalian cells [12]. Leishmania is an early diverging eukaryote whose genes are expressed constitutively as part of long polycistronic units where the RNAs are matured by coupled transsplicing and polyadenyl- ation (reviewed in [13]) and by epigenetic marks [14–16]. Gene regulation occurs mostly at the posttranscriptional and (post)trans- lational levels [13] with no control at the level of transcription initiation, in part due to the lack of several general transcription factors [17]. Leishmania display, however, additional strategies to modulate the expression of specific genes when selective pressure is applied. For example, Leishmania cells selected for resistance to PLOS Biology | www.plosbiology.org 1 May 2014 | Volume 12 | Issue 5 | e1001868