Comparative analyses of two primate species diverged by more than 60 million years show different rates but similar distribution of genome-wide UV repair events

Abstract

Background: Nucleotide excision repair is the primary DNA repair mechanism that removes bulky DNA adducts such as UV-induced pyrimidine dimers. Correspondingly, genome-wide mapping of nucleotide excision repair with eXcision Repair sequencing (XR-seq), provides comprehensive profiling of DNA damage repair. A number of XR-seq experiments at a variety of conditions for different damage types revealed heterogenous repair in the human genome. Although human repair profiles were extensively studied, how repair maps vary between primates is yet to be investigated. Here, we characterized the genome-wide UV-induced damage repair in gray mouse lemur, Microcebus murinus, in comparison to human.

Results: We derived fibroblast cell lines from mouse lemur, exposed them to UV irradiation, and analyzed the repair events genome-wide using the XR-seq protocol. Mouse lemur repair profiles were analyzed in comparison to the equivalent human fibroblast datasets. We found that overall UV sensitivity, repair efficiency, and transcription-coupled repair levels differ between the two primates. Despite this, comparative analysis of human and mouse lemur fibroblasts revealed that genome-wide repair profiles of the homologous regions are highly correlated, and this correlation is stronger for highly expressed genes. With the inclusion of an additional XR-seq sample derived from another human cell line in the analysis, we found that fibroblasts of the two primates repair UV-induced DNA lesions in a more similar pattern than two distinct human cell lines do.

Conclusion: Our results suggest that mouse lemurs and humans, and possibly primates in general, share a homologous repair mechanism as well as genomic variance distribution, albeit with their variable repair efficiency. This result also emphasizes the deep homologies of individual tissue types across the eukaryotic phylogeny.

Keywords: (6–4)PP; CPD; Mouse Lemur; Nucleotide excision repair; Primate; UV damage; XR-seq.

Fig. 1

Characteristics of excised oligomers upon repair of UV-induced damages. (A) In vivo excision assay for (6–4)PP and CPD are shown. (B) XR-seq read length distribution of excised oligomers for CPD (top) and (6–4)PP (bottom). Human and mouse lemur are shown in blue and red, respectively. (C) Nucleotide frequency in the predominant oligomer (26 nt) for each primate and damage type. Representative data from replicate 1

Fig. 2

UV sensitivity and repair rates in human and lemur fibroblasts. (A) Clonogenic survival assay (left) and the quantified results (right). (B) Immunoslot blot repair assays to examine the repair kinetics of UV-induced DNA lesions for both organisms and each damage type (left). Remaining damage levels at each time point were quantified and plotted (right). All experiments were repeated three times, and graphed data are presented as (mean+/−SD). Tests (t-test) performed (H₀:μ_human = μ_lemur) for each dosage (A) and time points (B). * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; rest P > 0.05

Fig. 3

Transcription-coupled repair in mouse lemur and human fibroblasts. Average profiles of CPD XR-seq (top) and (6–4)PP XR-seq (bottom) over 5277 and 3366 annotated genes are plotted for human (left) and mouse lemur (right), respectively. Transcription start sites (TSS) and transcription end sites (TES) were retrieved from GTF (Gene Transfer Format) files for the two genomes. Transcribed (red) and nontranscribed (blue) strand repair are shown in the downstream and upstream of TSS and TES, respectively. 10 kb upstream and downstream of TSS and TES were divided into 100 bp windows. XR-seq reads aligned to each bin were normalized to RPKM (reads per kilobase per million mapped reads). The XR-seq RPKM values were normalized by the shuffled RPKM values derived by mapped reads aligned to random genomic sites. Only replicate 1 is shown. TS/NTS median values for TSS downstream for CPD are 1.76 and 2.31 for human and mouse lemur, respectively

Fig. 4

Comparative analysis methodology and correlated repair rates between human and mouse lemur. A) Approach to identify orthologous regions between human and mouse lemur (see methods for details). B) Scatter plots showing the normalized repair levels between two organisms. Relative repair was calculated by normalizing the repair signal by the simulated XR-seq reads to eliminate the sequence context bias. C) Mapped orthologous regions with repair and transcription profiles mapped at left and right outer rings, respectively. Inner circle represents the human (left) and mouse lemur (right) chromosomes. Inner lines connecting orthologous regions are colored based on the human chromosome color scale. Repair and transcription values (outer rings) for genomic regions that have no evident ortholog (based on the criteria in A) are not shown

Fig. 5

Repair rate consistency in correlation with gene expression. The transcription levels of the orthologous regions were divided into quartiles separately for human and lemur. Out of the quartiles, 10 bootstrapped subsamples were retrieved, and for those regions repair correlation was analyzed. Correlation coefficient (R values) are represented on the y-axis; quartiles are on the x-axis

Fig. 6

Cell type-based consistency of repair profiles between two primates. A) Scatter plot or normalized repair levels between two human cell lines, GM18787 and NHF1, show correlation (R = 0.65, p = 0). B) Principle component analysis of 10 XR-seq samples