Single-molecule analysis of DNA replication reveals novel features in the divergent eukaryotes Leishmania and Trypanosoma brucei versus mammalian cells

Abstract

Leishmania and Trypanosoma are unicellular parasites that possess markedly original biological features as compared to other eukaryotes. The Leishmania genome displays a constitutive 'mosaic aneuploidy', whereas in Trypanosoma brucei, the megabase-sized chromosomes are diploid. We accurately analysed DNA replication parameters in three Leishmania species and Trypanosoma brucei as well as mouse embryonic fibroblasts (MEF). Active replication origins were visualized at the single molecule level using DNA molecular combing. More than one active origin was found on most DNA fibres, showing that the chromosomes are replicated from multiple origins. Inter-origin distances (IODs) were measured and found very large in trypanosomatids: the mean IOD was 160 kb in T. brucei and 226 kb in L. mexicana. Moreover, the progression of replication forks was faster than in any other eukaryote analyzed so far (mean velocity 1.9 kb/min in T. brucei and 2.4-2.6 kb/min in Leishmania). The estimated total number of active DNA replication origins in trypanosomatids is ~170. Finally, 14.4% of unidirectional replication forks were observed in T. brucei, in contrast to 1.5-1.7% in Leishmania and 4% in MEF cells. The biological significance of these original features is discussed.

Figure 1. Schematic representation of the experimental approach and different signal patterns observed after two-pulse labelling of replicating DNA.

(A) Asynchronous cells were successively labelled with equal pulses of iodo-deoxyuridine (IdU) and chloro-deoxyuridine (CldU) and chromosomal DNA combed on silanized coverslips. (B) Three major types of signal patterns may be observed after molecular combing and immuno-detection of the modified nucleosides, depending whether DNA replication initiates before the first pulse (Ori 1), during the first pulse (Ori 2) or during the second pulse (Ori 3). DNA fibres are counterstained in blue; IdU tracks, corresponding to the incorporation of the modified nucleotide during the first pulse, are revealed in red, whereas CldU tracks corresponding to the second pulse are in green. Arrows represent the direction of the replication forks progression, which is visually detected as a transition from red to green labeling. DNA replication initiation sites (active origins or Ori) stand in the centre of the bidirectional replication fork. The inter-origin distance (IOD) was determined by measuring centre to centre distance between two adjacent progressing bidirectional forks.

Figure 2. Single-molecule analysis of the inter-origin distances in trypanosomatids using molecular combing.

(A) Representative chromosome fibres from trypanosomatids used for the analysis of inter-origin distances (IOD). Positions of the presumed origins are indicated with arrows. Scale bar: 50 kb. For each species, upper panels: counterstaining of DNA fibres is in blue, first pulse nucleosides (IdU) in red and second pulse nucleosides (CldU) in green; lower panels show the tracks from the first and second pulse nucleosides extracted from the upper panel. (B) Statistical distribution (frequency) of IODs in MEF cells and four trypanosomatids. (C) Comparative analysis of IODs in MEF cells and four trypanosomatids obtained after one-way ANOVA analysis of variance. Boxes present 25–75% range. Whiskers present minimum–maximum range. The two-tailed Mann-Whitney test was used to compare the median values of IOD between each two samples and to calculate the corresponding P values (P < 0.05 was taken as significant).

Figure 3. Single-molecule analysis of the velocity of replication forks in MEF cells and four trypanosomatids using DNA molecular combing.

(A) Representative bidirectional replication forks taken from different microscopic fields were artificially assembled and centred on the position of the presumed origins. Red tracks: IdU, green tracks: CldU. Counterstaining of DNA was performed but is not shown here for the sake of clarity. Scale bar: 50 kb. (B) Statistical distribution of replication forks velocity measurements in MEF cells and four trypanosomatids. The velocity of the replication fork was calculated by dividing the length of the ‘green’ tracks with the duration of the second pulse. (C) Comparative analysis of the velocity of replication forks in the five cell lines obtained after one-way ANOVA analysis of variances. Boxes present 25–75% range. Whiskers present minimum–maximum range. The two-tailed Mann-Whitney test was used to compare the median values of the velocity of replication forks between each two samples and to calculate the corresponding P values (P < 0.05 was taken as significant).

Figure 4. Positive correlation between mean inter-origin distances (IODs) and fork velocities.

2-way representation of mean values of fork velocities and IODs in MEF cells, T. brucei and three Leishmania species.

Figure 5. Analysis of synchronous and asynchronous adjacent origin firing.

(A) Representative chromosome fibres used for the analysis of DNA replication dynamics showing synchronous (upper picture) and asynchronous (lower picture) firing of adjacent origins (arrows). Diagrams: “a” and “b” represent the lengths of the tracks of non-labelled DNA in two adjacent origins. They are used to discriminate between synchronous and asynchronous origins: if “a = b” the two adjacent origins are synchronous, if “a ≠ b”, they are asynchronous. (B) The number of DNA fibres bearing synchronous and asynchronous adjacent origin firing was counted in the five cell types (MEF cells, T. brucei and three Leishmania species). Numbers and percentages are presented in the table (right) and the percentages of each type of firing represented as histograms (left). The Chi-square test was used to calculate the P values (P < 0.05 was taken as significant).

Figure 6. Analysis of the symmetry of progressing bidirectional replication forks.

(A) Presentation of the concept of long fork/short fork ratios. The long fork/short fork ratio corresponds to the ratio of the longer IdU + CldU signal over the shorter IdU + CldU signal of bidirectional replication forks. A ratio >1 indicates fork asymmetry, while a ratio =1 indicates symmetry (see Materials & Methods). (B) Distribution of long fork/short fork ratios in MEF cells, T. brucei and three Leishmania species. Median (black bar) values are indicated below the distribution clouds. Statistically significant differences are indicated; p values were calculated using the two-tailed Mann-Whitney test. (C) Representative pairs of asymmetric replication forks from T. brucei. The forks were assembled from different microscope fields. Arrows indicates the lengths of the bidirectional forks analysed. Red tracks: IdU, green tracks: CldU, blue tracks: DNA. DNA counterstaining is not shown in the bottom panel. Scale bar: 50 kb. (D) Schematic presentation of the unidirectional replication forks that were analysed. Forks indicated by asterisks are counted as unidirectional forks and used in the calculation of percentage of unidirectional forks. In the top fibre, the left fork was not included for calculation as one cannot distinguish between a unidirectional fork and a fibre broken between the bidirectional forks. (E) Histograms showing the percentage of unidirectional replication forks over the total number of analysed forks in MEF cells, T. brucei and three Leishmania species. (F) Representative DNA fibres from T. brucei showing unidirectional forks. The fibres were assembled from different fields. Arrows indicate the direction of replication forks. Unidirectional forks indicated by asterisks were used for this analysis. Same color code and legend as in (C).