- Results: Here we have mapped sites of DNA replication initiation across the T. - Specific DNA sequences to define the replicator are found in the ge- nomes of bacteria, some archaea [2], and in Saccharomy- ces cerevisiae and related species [3]. - Instead, association of the initiator with origins may be dictated by nuclear architecture, gene density, chromatin status (such as histone modification or nucleosome posi- tioning), transcriptional activity, and AT or CG content [4–6].. - In eukaryotes, the initiator is termed the Origin Recog- nition Complex (ORC) [7, 8] and is assembled at replica- tion origins during mitosis-G1 phases of the cell cycle, when it recruits, via Cdc6 and Cdt1, the MCM replica- tion helicase, allowing origins become ‘licensed’ [9, 10].. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - 1 Laboratório de Ciclo Celular, Instituto Butantan, São Paulo, Brazil Full list of author information is available at the end of the article. - and stages of development, and even in different cells of the same population. - Collision between replication and transcription is considered especially problematic, as each are catalyzed by large multiprotein machines, and can occur co-directionally, when the replication fork and transcription machinery are moving in the same direc- tion, or on the leading strand and is head-on, when the fork and transcription are moving towards each other.. - brucei origins are found at the boundaries of the DGCs, which all appear to bind ORC1/CDC6. - Nonetheless, there are connections between transcription and replication: MFA-seq data shows DNA replication is more strongly impeded as it meets tran- scription head-on [23], RNAi of ORC1/CDC6 increases transcript abundance at the start and end of the DGCs (Tiengwe et al., 2012), telomere transcription levels in- fluence replication timing, and telomere variation is dependent on ORC1/CDC6 levels [25]. - brucei and Leish- mania was used to extrapolate a greater number of pre- dicted sites of DNA replication initiation than origins mapped by MFA-seq, though was not able determine if these predicted sites relate to ORC binding or, indeed, in what part of the genome they might reside [28]. - 5000 sites of DNA replication initiation, with very limited correlation with the ends of the DGCs [29].. - Taken as a whole, these studies might indicate that complete replication of the entire genome in the rela- tively short S phases of T. - de Araujo et al. - A limitation of the emerging data discussed above is that so far only two members of the trypanosomatid grouping have been examined, meaning the basis for the differences in DNA replication in T. - Nonethe- less, in the last years, evidence has accumulated suggest- ing that gene arrays are sites that favor homologous recombination (HR) [36–38] as the driver of genetic variability among these families [38]. - Despite the technical challenge of next generation se- quence mapping in this uniquely repetitive genome, we show that some sites of replication initiation map to the borders of the DGCs, as seen for MFA-seq mapping in T. - cruzi DNA replication origins were determined by two different approaches. - In order to investigate DNA replication dynamics in T.. - Two hundred forty seven putative initiation sites were predicted in the P haplotype of R1 (Table S3) and 247 of R2 (Table S4). - 234 in the S haplo- type of R1 (Table S5) and 235 of R2 (Table S6). - Also, when we found two peaks in one replicate but only one in another replicate, we se- lected just the peak closer to the one of the first repli- cate. - In the end, 129 MFA-seq peaks pair were compared (Table S7): the median distance between them. - This approach predicted 304 and 260 pu- tative replication initiation sites, respectively, in the P haplotype R1 (Table S3) and S haplotype R1 (Table S5).. - 2a and b, with 110 and 103 consensus replication initiation sites common to the two approaches in the P (Table S8) and S haplotypes (Table S9). - We next analyzed the genomic location of the S/G2 enriched regions, comparing the location of peaks esti- mated by fold change analysis, the consensus peaks (representing intersection between fold change and MACS2), GC content and transcription orientation across all T. - 1 Mapping replication origins in the T. - Graphs show the extent of DNA enrichment in S phase relative to G2, in the indicated chromosomes. - The intersection between them corresponds to the consensus of the peaks, which have been determined as origins of replication. - Therefore, we decided to analyse the presence of ORIs in the entire genome according to distribution of MGF. - Although we found ORIs in both conserved and disrupted compart- ments, there is an enrichment of ORIs in the disrupted region (Additional file 2).. - In addition, the CG content of DGF-1 ORIs is 68.25% (S) and 68.20% (P), while the CG content in the rest of identi- fied ORIs is 62.36% (S) and 60.14% (P).. - We note in the Additional file 1 that some chromo- somes seem to present putative ORI at the same location in both P and S haplotypes (see chromosomes as examples), while in others, the location changes when compared to different haplotypes. - 5: Chromosomes 3 and 6 that, apparently, contain ORIs in the same position in both haplotypes. - In the graphs also depicted the GC content (GC) along the chromosome and the directional gene clusters (CDS). - This is evi- dent on chromosome 6 where putative ORIs are in the same location, haplotypes are sythenic but the specific region that contains ORI is not synthenic. - How- ever, since even in the syntenic regions of S and P haplo- types there are differences between the homologous chromosomes due to the duplication/deletion events oc- curred during T. - Strikingly, there was a clear correlation between replication initiation site location (beginning or end of chromosome as annotated in the trytripdb.org) and transcription orientation: more replication initiation sites were located at the beginning of chromosomes in regions where transcription is also orientated towards the chro- mosomes’ beginning (negative strand, Fig. - The graphs show the percentage of AT and GC contents in all 41 chromosomes of the Esmeraldo-like (a) and Non-Esmeraldo-like (b) haplotypes. - Central solid lines correspond to the contents of the AT and GC bases found in the T. - To further investigate the predicted replication initiation sites in the T. - Like in the P haplotype, most of the putative. - Since DGF-1 genes repre- sent only 3.33% of the total genome in base pairs on both haplotypes (Table S14) and are not the most. - Genes transcribed in the negative strand. - genes transcribed in the positive strand. - Nineteen putative origins in the P haplotype and 19 in S (Table S15) matched with previ- ously annotated subtelomeric regions. - In performing this analysis, we found that the frequency of SNPs was highest towards the periphery of the chro- mosomes in both haplotypes (Fig. - Next, we com- pared the frequency of SNPs in DGF-1 genes predicted to contain putative origins with those without predicted origins: SNPs were much abundant in the former (Fig.. - The number of puta- tive ORIs obtained either by fold change or by MACS2 is higher than obtained after prediction of the consensus peaks suggesting that using the consensus approach might have a more reliable set by excluding false positive ORIs. - cerevisiae, origins are enriched with AT- se- quences but in the other eukaryotes, origins are GC-rich sequences [44], including CpG islands and G-rich ele- ments of the Origin G-rich repeated element (OGRE), which have great potential to form secondary DNA con- formations, such as G-quadruplex [45, 46], and interca- lated motif (i-motif) [47, 48].. - cruzi CL Brener suggested a median inter-origin distance of 171.1 kb [41], which can be extrapolated to a total of 85 origins, which is close to 103 and 110 putative con- sensus origins mapped in the P and S haplotypes in this analysis. - In fact, the orientation of transcription to- wards chromosomes telomeres is a feature of the T.. - cruzi genome (Additional file 7), suggesting the greater abundance of putative origins in subtelomeres generates head-on transcription-replication collisions as the repli- somes move towards the centers of the chromosomes.. - avoid conflicts between transcription and replication machineries, such as temporal and special separation of transcription and replication processes, avoid stalled RNA polymerases in the genome, and orientation of highly transcribed genes in the same direction of replica- tion fork movement [49]. - Even organisms that present genes organized in operons (in the case of C. - brucei), origins are located in tran- scription start sites warranting the replication fork movement in the same direction than transcription [23, 50]. - In other words, subtelomeric replication initiation, where transcription occurs towards chromosome ends, may be at least one of the sources of DNA breaks, because transcription has been shown to arrest replication fork progression [51, 52]. - Interestingly, the majority of ORIs identified in this analysis was preferentially positioned inside the CDSs, mainly in the DGF-1gene. - It is known that many mem- bers of the DGF-1 gene family are located in subtelo- meric regions, where they may be prone to variability [56]. - It has been argued that trypanosomatids limit the presence of ORC-defined origins to SSRs, leading to widely spaced origins, in order to limit binding of the initiator and thereby limiting transcription-replication clashes in the context of multigenic transcription . - We envisage a similar event could occur in the replicative T. - We need to explore, for instance, whether (and which) DGF-1 is transcribed at S phase of replicative forms and if these DGF-1 transcribed at replicative forms would be expressed in the infective forms where the larger repertoire of DGF-1 isoforms could contribute for infection. - Though we cannot be cer- tain the regions of increased S/G2 enrichment truly rep- resent origins, since we have not mapped localization of the ORC machinery, our data suggest that while some T.. - Further work is also warranted to ask what features of the T. - The S and G2 phase paired-reads were aligned against the genomic sequences of the Esmeraldo-like and non Esmeraldo-like haplotypes using the software Bowtie2 v with the parameters “end- to-end” whole paired-read alignment (−-very-sensitive), reporting only the best alignment (−k 1) for paired-reads. - Similarly, were used S and G2 data to estimate peaks of enriched regions in the genome using ‘macs2 callpeak’ function from MACS2 software, which uses a Poisson distribution to calculates a dy- namic Poisson parameters for each region to obtain a distri- bution having more flexibility than the negative binomial distribution [64]. - The datasets generated and/or analyzed during the current study are available in the NCBI BioProject repository, under the accession code PRJNA635749 (MFA-seq bioproject) and BioSample codes SAMN15052360 (Epimastigote at G2 stage, replicate 1), SAMN15052361 (Epimastigote at G2 stage, rep- licate 2), SAMN15052345 (Epimastigote at S stage, replicate 1), and SAMN15052356 (Epimastigote at S stage, replicate 2) (see Table 2 for details).. - Each one of the chromosomes from P and S haplotypes were binned into 1083 bp sections, and the average value across each bin was used to calculate the mean and the standard deviation of GC- content for each chromosome, considering the whole chromosome. - 40.0 - the Root Mean Square of the mapping quality of the reads across all samples. - 8.0 - the u- based z-approximation from the Mann-Whitney Rank Sum Test for the distance from the end of the read for reads with the alternate allele).. - In the graphs also depicted the GC content (c) along the chromosome and the directional gene clusters (d). - Clusters of CDSs that are transcribed in the same direction represent a DGC. - genes transcribed in the positive strand.. - Total number of raw paired-end reads for each sample and respective replicate, followed by the high quality paired-end reads after the pre-processing quality approach and the per- centage of mapped paired-end reads in the CL Brenner EL and NEL gen- ome haplotypes. - Percentage of DGF-1(bp) in the entire genome Table S15. - S: Esmeraldo-like. - The authors thank the Program for Technological Development in Tools for Health-RPT-FIOCRUZ for the use of the flow cytom- etry facility (RPT08L) at Carlos Chagas Institute — Fiocruz/PR. - Work in the UK was supported by the BBSRC [BB/N016165/1], the Wellcome Trust Fundação para a Ciência e Tecnologia [SFRH/BD and the European commission (through a Marie Curie fellowship to JD [750259, RECREPEMLE]).. - The reference genome used in the present work is available at TriTrypDB release 32, version CLBrenerNonEsmeraldo-like: https://tritrypdb.. - The datasets generated and/or analyzed during the current study are available in the NCBI BioProject repository, under the acces- sion code PRJNA635749 and BioSample codes SAMN15052360,. - On the regulation of DNA replication in Bacteria. - DNA replication origins in archaea. - DNA replication origins-where do we begin?. - Regulating DNA replication in eukarya. - Conflict resolution in the genome: how transcription and replication make it work. - Chromatin conformation regulates the coordination between DNA replication and transcription. - Report of the Tenth Meeting of the WHO Strategic and Technical Advisory Group for Neglected Tropical Diseases. - Cell biology of the trypanosome genome.. - Diverged composition and regulation of the Trypanosoma brucei origin recognition complex that mediates DNA replication initiation. - Genome-wide analysis reveals extensive functional interaction between DNA replication initiation and transcription in the genome of Trypanosoma brucei. - Single molecule analysis of Trypanosoma brucei DNA replication dynamics. - Single- molecule analysis of DNA replication reveals novel features in the divergent eukaryotes Leishmania and Trypanosoma brucei versus mammalian cells. - Transcriptionally driven DNA replication program of the human parasite Leishmania major. - Anatomy and evolution of telomeric and subtelomeric regions in the human protozoan parasite Trypanosoma cruzi. - Recombination-driven generation of the largest pathogen repository of antigen variants in the protozoan Trypanosoma cruzi. - A phylogenetic analysis of the Trypanosoma cruzi genome project CL Brener reference strain by multilocus enzyme electrophoresis and multiprimer random amplified polymorphic DNA fingerprinting. - I- motif DNA structures are formed in the nuclei of human cells. - The conflict between DNA replication and transcription. - Biological parameters and molecular markers of clone CL Brener--the reference organism of the Trypanosoma cruzi genome project
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