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. 2020 Dec 29;24(1):102005. doi: 10.1016/j.isci.2020.102005

Disproportionate presence of adenosine in mitochondrial and chloroplast DNA of Chlamydomonas reinhardtii

Waleed MM El-Sayed 1,2,3, Alli L Gombolay 1,7, Penghao Xu 1,7, Taehwan Yang 1,7, Youngkyu Jeon 1,7, Sathya Balachander 1,7, Gary Newnam 1, Sijia Tao 4, Nicole E Bowen 4, Tomáš Brůna 1, Mark Borodovsky 1,5,6, Raymond F Schinazi 4, Baek Kim 4, Yongsheng Chen 2, Francesca Storici 1,8,∗
PMCID: PMC7809514  PMID: 33490913

Summary

Ribonucleoside monophosphates (rNMPs) represent the most common non-standard nucleotides found in the genome of cells. The distribution of rNMPs in DNA has been studied only in limited genomes. Using the ribose-seq protocol and the Ribose-Map bioinformatics toolkit, we reveal the distribution of rNMPs incorporated into the whole genome of a photosynthetic unicellular green alga, Chlamydomonas reinhardtii. We discovered a disproportionate incorporation of adenosine in the mitochondrial and chloroplast DNA, in contrast to the nuclear DNA, relative to the corresponding nucleotide content of these C. reinhardtii organelle genomes. Our results demonstrate that the rNMP content in the DNA of the algal organelles reflects an elevated ATP level present in the algal cells. We reveal specific biases and patterns in rNMP distributions in the algal mitochondrial, chloroplast, and nuclear DNA. Moreover, we identified the C. reinhardtii orthologous genes for all three subunits of the RNase H2 enzyme using GeneMark-EP + gene finder.

Subject areas: Molecular Biology, Genomics

Graphical Abstract

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Highlights

  • •

    The sites of ribonucleotides embedded in Chlamydomonas reinhardtii DNA are revealed

  • •

    Adenosine is disproportionally abundant in algal mitochondrial and chloroplast DNA

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    There is a high ATP level present in the algal cells

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    C. reinhardtii orthologous genes for the three subunits of ribonuclease H2 are found

Molecular Biology; Genomics

Introduction

The presence of ribose sugar in place of deoxyribose in DNA is a common DNA modification due to the abundant incorporation of ribonucleoside monophosphates (rNMPs), which are the units of RNA, by DNA polymerases (Nava et al., 2020; Williams et al., 2016). While it has been known for a long time that rNMPs are present in specific DNA sequences, such as mouse and human mitochondrial DNA (Grossman et al., 1973), at the mating type locus in the nuclear DNA of fission yeast (Vengrova and Dalgaard, 2006) and even in chloroplast DNA (Kolodner et al., 1975), only in the last decade has the ribose in DNA been defined as the most abundant alteration in the DNA of cells (Caldecott, 2014; Cavanaugh et al., 2010; Clausen et al., 2013; Gosavi et al., 2012; Kasiviswanathan and Copeland, 2011; Kennedy et al., 2012; Lemor et al., 2018; McDonald et al., 2012; Nick McElhinny et al., 2010; Potenski and Klein, 2014; Williams and Kunkel, 2014; Williams et al., 2016). Recent studies highlight the capacity of many DNA polymerases to incorporate rNMPs into DNA (Astatke et al., 1998; Bonnin et al., 1999; Brown and Suo, 2011; Cavanaugh et al., 2010; Gong et al., 2005; Kasiviswanathan and Copeland, 2011; Kennedy et al., 2012; McDonald et al., 2012; Nick McElhinny and Ramsden, 2003; Patel and Loeb, 2000). For example, Escherichia coli polymerase V (McDonald et al., 2012), the polymerase component of bacterial non-homologous end joining ligases (Zhu and Shuman, 2008), all replicative polymerases of budding yeast (Pol α, δ, and ε) (Nick McElhinny et al., 2010), and the human replicative polymerase δ (Clausen et al., 2013) can insert rNMPs into DNA. Human DNA polymerases λ and μ can insert rNMPs with the same efficiency as deoxyribonucleoside monophosphates (dNMPs) (Gosavi et al., 2012; Moon et al., 2017). In addition, the reverse transcriptase of the human immunodeficiency virus inserts 1 rNMP per 146 dNMPs in the viral genome before integrating into human macrophage DNA (Kennedy et al., 2012). Although these data suggest that rNMPs in DNA are broadly present in nature, studies examining primarily the positions, patterns, and hotspots of rNMPs in DNA have only been done for yeast genomic DNA (Balachander et al., 2020; Clausen et al., 2015; Daigaku et al., 2015; Jinks-Robertson and Klein, 2015; Koh et al., 2015; Reijns et al., 2015). Because ribonuclease (RNase) H2 is the major enzyme removing rNMPs from DNA (Sparks et al., 2012), the initial mapping of rNMPs in DNA to a single-nucleotide resolution was done in yeast strains with non-functional RNase H2 (Clausen et al., 2015; Daigaku et al., 2015; Jinks-Robertson and Klein, 2015; Koh et al., 2015; Reijns et al., 2015). Moreover, several reports have shown that RNase H2 is not active in yeast and human mitochondrial DNA (Balachander et al., 2020; Berglund et al., 2017; Wanrooij et al., 2017). This likely explains why, using a collection of Saccharomyces cerevisiae mutants with altered nucleotide pool composition, it was shown that variations in the deoxynucleotide triphosphate (dNTP) pool can significantly affect the rNMP-incorporation frequency of mitochondrial DNA either in wild-type or in RNase H2-defective cells, but not that of nuclear DNA in wild-type RNase H2 cells (Wanrooij et al., 2017). Similarly, depletion of dNTP pools promoted rNMP incorporation in human mitochondrial DNA (Berglund et al., 2017). While nucleotide pool imbalances certainly play a major role in rNMP composition in genomic DNA, there are factors beyond variation in nucleotide pools that affect distribution and patterns of rNMP incorporation in DNA. Recently, by studying rNMP profiles in the genomes of three yeast species (S. cerevisiae, Saccharomyces paradoxus, and Schizosaccharomyces pombe) and several different strains of these yeasts having either wild-type or mutant RNase H2, we showed low levels of rU in the mitochondrial DNA as well as in the nuclear DNA of RNase H2-defective cells of all three yeast species, as well as dominant rC and low rG in the nuclear DNA of wild-type RNase H2 cells of all three yeast species (Balachander et al., 2020). We further observed non-uniform distribution in both the yeast nuclear and mitochondrial DNA, uncovering that the dNMP immediately upstream of the rNMP has a strong impact on the pattern of rNMP incorporation in mitochondrial as well as nuclear yeast DNA (Balachander et al., 2020).

The inclusion of rNMPs in DNA alters its stability, structure, plasticity, and ability to interact with proteins (Chiu et al., 2014; Klein, 2017). The presence of rNMPs in DNA may also regulate/modulate cellular functions and if conserved could manifest some type of epigenetic interaction in the cell. Thus, it is important to map rNMP sites in DNA and to characterize their features and rules of incorporation to understand the biological significance of rNMPs in DNA and determine whether these features and rules are conserved across different organisms or cell types. Our molecular and computational approaches, ribose-seq (Balachander et al., 2019) and Ribose-Map (Gombolay et al., 2019), allow for the efficient construction and analysis of genomic libraries derived from any DNA source containing rNMPs. Exploiting these techniques, we focused on the unicellular green alga of the species Chlamydomonas reinhardtii, which is broadly distributed worldwide in soil and freshwater. C. reinhardtii is used in production of biofuels (Sasso et al., 2018). It is also used to study photosynthesis and cell mobility (Sasso et al., 2018). We built ribose-seq libraries of rNMP incorporation from three independent cultures of C. reinhardtii cells grown in the light. We found a strongly biased frequency of incorporation of rAMP in the mitochondrial and chloroplast DNA, and we characterized the overall genomic rNMP distribution in these algal cells. Moreover, to characterize the capacity of C. reinhardtii to process rNMPs embedded in DNA, we identified genes and proteins of RNase H2 in the alga with help of the GeneMark-EP+ pipeline (Bruna et al., 2020).

Results

C. reinhardtii cells have a high ATP/dATP ratio

With the goal to study the composition and distribution of rNMPs in the DNA of C. reinhardtii cells, we first determined the concentration of nucleotide triphosphates (NTPs) and dNTPs in these algal cells. C. reinhardtii (strain CC-1690) cells were grown in the light for 5–7 days to reach optical density (O.D.) = 1.5–1.7 at 750 nm corresponding to 2×106 cells/mL. Cells were lysed, and cell extracts were prepared for mass spectrometry analyses of the NTP (ATP, CTP, GTP, and UTP) and dNTP (dATP, dCTP, dGTP, and dTTP) pools. The mass spectrometry analyses revealed strong abundance of ATP in the C. reinhardtii cells (Figure 1A). While NTPs are generally more abundant than dNTPs in cells, with NTP concentrations being one to three orders of magnitude higher than those of dNTPs (Clausen et al., 2013; Ferraro et al., 2010), a key factor contributing to misincorporation of rNMPs in DNA is a variation in nucleotide pool concentrations resulting in an increased NTP/dNTP ratio for one or more nucleotides (Ferraro et al., 2010; Wanrooij et al., 2017). In C. reinhardtii cells, the ATP/dATP ratio was by far the largest of the NTP/dNTP ratios in these cells (Figure 1B) and significantly higher than all other NTP/dNTP ratios (CTP/dCTP, GTP/dGTP, and UTP/dTTP, p = 0.0007, 0.004, and 0.0006, respectively). Compared to the previously recorded ATP/dATP ratios obtained for yeast cells, varying from ~100 to ~300 in S. cerevisiae and ~750 in S. pombe (Balachander et al., 2020; Clausen et al., 2013) and between ~130 and ~350 in human dividing cells and ~1,450 in human non-dividing cells (Clausen et al., 2013; Ferraro et al., 2010; Traut, 1994), the ATP/dATP ratio that we measured in C. reinhardtii cells is the highest, ~1,800 (Figure 1B). To determine whether the high ATP/dATP ratio observed in C. reinhardtii cells was due to the exposure to the light during cell growth, we measured the NTP and dNTP concentrations from algal cells that were grown in the dark for 10–12 days to reach O.D. = 1.5–1.7 at 750 nm corresponding to 2×106 cells/mL. While we found a slight decrease of the ATP/dATP ratio for cells grown in the dark, this ratio remained high and the largest compared to the other NTP/dNTP ratios (Figure S1). These results suggest that the high ATP/dATP ratio is a feature of the algal C. reinhardtii cells and it is independent from photosynthesis.

Figure 1.

Figure 1

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Measurements of NTP and dNTP levels in C. reinhardtii cells show high ATP concentration

(A) The levels of NTPs and dNTPs extracted from known numbers of algal cells of C. reinhardtii strain CC-1690 were determined by lliquid chromatography with tandem mass spectrometry (LC-MS-MS) methods (see Methods). BLOQ: below limit of quantification. The cellular level of each nucleotide was normalized for 107 cells.

(B) The NTP and dNTP levels determined in (A) were used to calculate the NTP/dNTP ratios. Shown are mean and standard deviation of four repeats for each sample. Ratios for dCTP and dTTP levels below the limit of quantification were calculated by using the limit value of quantification for dCTP or dTTP (0.02 pmole) relative to the number of cells used in the LC-MS/MS method. The ratio of ATP/dATP was clearly significantly higher than all other NTP/dNTP (CTP/dCTP, GTP/dGTP, and UTP/dTTP) ratios, p = 0.0007, 0.004, and 0.0006, respectively.

Mitochondrial and chloroplast DNA of C. reinhardtii have overriding incorporation of rA

To determine the pattern of rNMPs in the mitochondrial, chloroplast, and nuclear genome of C. reinhardtii cells, we extracted the whole genomic DNA from three independent cultures of these cells grown in light (see Transparent methods). From these three DNA extracts, we constructed three genomic libraries using the ribose-seq approach (see Transparent methods and Table S1): FS121, FS231, and FS232 (Table S2). Each of these ribose-seq libraries was sequenced and then segmented into a mitochondrial, chloroplast, and nuclear library using the Ribose-Map computational toolkit after alignment of the sequencing reads to the reference genome sequence of C. reinhardtii. We obtained the mitochondrial and chloroplast sequences from the NCBI (https://www.ncbi.nlm.nih.gov/genome/147) and the 5.5 nuclear sequence from the Joint Genome Institute (JGI) (https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Creinhardtii). The percentage of rNMPs with base A, C, G and U (rA, rC, rG, and rU) varies strikingly between ribose-seq libraries of the organelles and nuclear DNA. Remarkably, both the mitochondrial and chloroplast DNA display a noticeable preference for rA with an average of 89% rA in mitochondrial and 77% rA in chloroplast DNA (Figure 2 and Table S2). As shown by our analysis of the rNMP content in mitochondrial and chloroplast DNA of C. reinhardtii cells, rA is significantly and disproportionally incorporated relative to rC, rG, and rU and relative to the nucleotide content of these genomes (Figures 3A–3D, 4A, and 5A and Tables S3 and S3B). The biased incorporation of rA in these organelles likely reflects the high ratio of ATP/dATP in the algal cells (Figure 1B). Interestingly, while rC, rG, and rU are all similarly infrequent in the mitochondrial DNA, rU is distinctly the least abundant rNMP in the chloroplast DNA, even if rC and rG are much less frequent than rA, as evidenced via nucleotide frequency and heatmap analyses (Figures 3A–3D, 4A, and 5A, and Tables S3 and S3B). The rNMP composition found in mitochondrial and chloroplast DNA of C. reinhardtii is maintained similarly between the forward and reverse strands of these genomes (Table S2).

Figure 2.

Figure 2

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High frequency of rA in mitochondrial and chloroplast but not nuclear genome of C. reinhardtii cells

Bar graph with the percentage of rA, rC, rG, and rU found in mitochondrial, chloroplast, and nuclear DNA of C. reinhardtii cells. Mean and standard deviation from three different mitochondrial, chloroplast, or nuclear ribose-seq libraries are shown.

Figure 3.

Figure 3

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Identity and sequence context of rNMPs in mitochondrial, chloroplast, and nuclear DNA of C. reinhardtii cells

Zoomed out (A, C, and E) and zoomed in (B, D, and F) plots of normalized nucleotide frequencies relative to mapped positions of sequences from mitochondrial (A and B), chloroplast (C and D), and nuclear (E and F) ribose-seq libraries. Position 0 is the rNMP; - and + positions are upstream and downstream dNMPs, respectively, normalized to the A, C, G, and T content in the genome of C. reinhardtii. The y axis shows the frequency of each type of nucleotide present in the ribose-seq data normalized to the frequency of the corresponding nucleotide present in the reference genome of the indicted cell compartment of C. reinhardtii. Red square, A; blue circle, C; orange triangle, G; and green rhombus, U. Red square, A; blue circle, C; orange triangle, G; and green rhombus, U.

Figure 4.

Figure 4

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rA is by large the most abundantly incorporated rNMP in C. reinhardtii mitochondrial DNA

(A) Heatmap analyses with (left) ratio of each type of rNMP (rA, rC, rG, and rU) and (right) ratio of each type of rNMP normalized to the nucleotide frequencies of the C. reinhardtii mitochondrial reference genome for the mitochondrial ribose-seq libraries of this study. The corresponding formulas used are shown in (B) and explained in the Transparent methods. Each column of the heatmap shows results of a specific ribose-seq library. Each library name is indicated underneath each column of the heatmap. Each row shows results obtained for an rNMP (R in red) of base A, C, G, or U for each library. The actual percentage of A, C, G, and T bases present in mitochondrial DNA of C. reinhardtii is shown to the left of the heatmap with normalized data. The bar to the right shows how different ratio values are represented as different colors. Black corresponds to 0.25.

(B) Heatmap analyses with normalized frequency of mitochondrial NR (top) and RN (bottom) dinucleotides containing rA with the upstream (top) or downstream (bottom) deoxyribonucleotide with base A, C, G, or T for the mitochondrial ribose-seq libraries of this study. The formulas used to calculate these normalized frequencies are shown and explained in the Transparent methods. Each column of the heatmap shows results of a specific ribose-seq library. Each library name is indicated underneath each column of the heatmap. Each row shows results obtained for a dinucleotide NR or RN (R in red) of fixed rNMP base A for each library. The actual percentage of dinucleotides of fixed base A for the indicated base combinations (AA, CA, GA, and TA, top; and AA, AC, AG, and AT, bottom) present in mitochondrial DNA of C. reinhardtii is shown to the left of the corresponding heatmaps. The observed percentage of dinucleotides with rA was divided by the actual percentage of each dinucleotide with fixed base A in mitochondrial DNA of C. reinhardtii. The bar to the right shows how different frequency values are represented as different colors. Black corresponds to 0.25. Significance of comparisons for data in this figure is shown in Table S3A.

Figure 5.

Figure 5

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rA is dominant and not uniformly distributed in C. reinhardtii chloroplast DNA

(A) Heatmap analyses with (left) ratio of each type of rNMP (rA, rC, rG and rU) and (right) ratio of each type of rNMP normalized to the nucleotide frequencies of the C. reinhardtii chloroplast reference genome for the chloroplast ribose-seq libraries of this study. The corresponding formulas used are explained in the Transparent methods. Each column of the heatmap shows results of a specific ribose-seq library. Each library name is indicated underneath each column of the heatmap. Each row shows results obtained for an rNMP (R in red) of base A, C, G, or U for each library. The actual percentage of A, C, G, and T bases present in chloroplast DNA of C. reinhardtii is shown to the left of the heatmap with normalized data. The bar to the right shows how different ratio values are represented as different colors. Black corresponds to 0.25.

(B) Heatmap analyses with normalized frequency of chloroplast NR (top) and RN (bottom) dinucleotides containing rA with the upstream (top) or downstream (bottom) deoxyribonucleotide with base A, C, G, or T for the chloroplast ribose-seq libraries of this study. The formulas used to calculate these normalized frequencies are shown and explained in the Transparent methods. Each column of the heatmap shows results of a specific ribose-seq library. Each library name is indicated underneath each column of the heatmap. Each row shows results obtained for a dinucleotide NR or RN (R in red) of fixed rNMP base A for each library. The actual percentage of dinucleotides of fixed base A for the indicated base combinations (AA, CA, GA, and TA, top; and AA, AC, AG, and AT, bottom) present in chloroplast DNA of C. reinhardtii is shown to the left of the corresponding heatmaps. The observed percentage of dinucleotides with rA was divided by the actual percentage of each dinucleotide with fixed base A in chloroplast DNA of C. reinhardtii. The bar to the right shows how different frequency values are represented as different colors. Black corresponds to 0.25. Significance of comparisons for data in this figure is shown in Table S3B.

Nuclear DNA of C. reinhardtii has higher level of rG and rC and lower level of rA and rU

The nuclear rNMPs were uniformly distributed across the nuclear genome with the percentage of rNMPs being proportional to the C. reinhardtii chromosome sizes (Figure S2). The composition of rNMPs in nuclear DNA showed more abundant rG and rC, followed by rA (Figures 2, 3E, 3F, and 6A, and Table S2). rU was consistently the least abundant rNMP in the nuclear DNA of the three libraries and on average a factor of ~2.8 and up to a factor of 4.7 below the nuclear-dT content (Figures 3E, 3F, and 6A, and Tables S2 and S3). As for mitochondrial and chloroplast DNA, the rNMP composition found in nuclear DNA of C. reinhardtii is maintained similarly between the forward and reverse strands of these genomes (Table S2). Normalization of single rNMP frequencies to the nucleotide base content of C. reinhardtii nuclear DNA revealed rNMP incorporation with a marked preference for rG and/or rC in all the nuclear libraries over rA and especially over rU (Figure 6A and Table S3). These nuclear data do not reflect the nucleotide pool composition of C. reinhardtii cells because, as shown above, among the measured ratios of NTPs/dNTPs in the algal cells, the ATP/dATP is the highest one (Figure 1B). These results suggest possible active removal of rA from nuclear DNA but not from the mitochondrial and chloroplast genomes.

Figure 6.

Figure 6

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rC and rG are dominant and not uniformly distributed in nuclear DNA of C. reinhardtii cells

(A) Heatmap analyses with (left) ratio of each type of rNMP (rA, rC, rG, and rU) and (right) ratio of each type of rNMP normalized to the nucleotide frequencies of the C. reinhardtii nuclear reference genome for the nuclear ribose-seq libraries of this study. The corresponding formulas used are shown in the Transparent methods. Each column of the heatmap shows results of a specific ribose-seq library. Each library name is indicated underneath each column of the heatmap. Each row shows results obtained for an rNMP (R in red) of base A, C, G, or U for each library. The actual percentage of A, C, G, and T bases present in nuclear DNA of C. reinhardtii is shown to the left of the heatmap with normalized data. The bar to the right shows how different ratio values are represented as different colors. Black corresponds to 0.25.

(B) Heatmap analyses with normalized frequency of nuclear NR (top) and RN (bottom) dinucleotides with rA, rC, rG, and rU with the upstream (left) or downstream (right) deoxyribonucleotide with base A, C, G, or T for the nuclear ribose-seq libraries of this study. The formulas used to calculate these normalized frequencies are shown and explained in the Transparent methods. Each column of the heatmap shows results of a specific ribose-seq library. Each library name is indicated underneath each column of the heatmap. Each row shows results obtained for a dinucleotide NR or RN (R in red) of fixed base A, C, G, or T for the indicated base combinations for each library. The actual percentage of dinucleotides of fixed base A, C, G, or T present in nuclear DNA of C. reinhardtii is shown to the left of the corresponding heatmaps. The observed percentage of dinucleotides with rNMPs with base A, C, G, or U was divided by the actual percentage of each dinucleotide with fixed base A, C, G, or T in nuclear DNA of C. reinhardtii. The bar to the right shows how different frequency values are represented as different colors. Black corresponds to 0.25. Significance of comparisons for data in this figure is shown in Table S3C.

Evaluation of RNase H2 activity in the algal cells and identification of the genes coding for the three subunits of RNase H2 in the C. reinhardtii genome

To examine whether the C. reinhardtii cells of strain CC-1690 displayed RNase H2 activity or other similar activity on a DNA substrate containing an embedded rNMP, protein extracts (Figure S3) were prepared to examine their activity to cleave a substrate with one rG or one rA (see Transparent methods). Cleavage of these substrates by E. coli RNase HII was used as a positive control. The results show that C. reinhardtii protein extract is not active on an rG in DNA because the dsDNA substrate was not cleaved at the rNMP position (Figure S3). Similar results were obtained using the DNA substrate containing rA (Figure S3). No active cleavage of rNMP-containing substrates from the C. reinhardtii protein extract was detected. It is possible that the RNase H2 activity in C. reinhardtii cells is weak. At the same time, we cannot exclude the possibility that the protein extract is either missing some cofactor necessary for the RNase H2 function of C. reinhardtii or it may contain some inhibitory factor for RNase H2, preventing RNase H2 from cutting the rNMP-containing substrates. When we treated the substrate containing the rG with E. coli RNase HII both in the presence and the absence of the algal protein extract, we noticed a modest decrease of RNase HII cleavage activity in the presence of the algal protein extract, although cleavage at the rG was still efficient (Figure S3).

To identify genes for RNase H2 subunits A, B, and C in the genome of C. reinhardtii, we used gene finder GeneMark-EP+ (Bruna et al., 2020). For finding gene for the subunit A, it was sufficient to run a component of GeneMark-EP+, the protein mapping ProtHint pipeline that detected relationship between the RNase H2 subunit A family of proteins and a gene in the C. reinhardtii chromosome 17 in positions 754,797–758,398, encoding a protein apparently homologous to 915 out of 1,059 target RNase H2 subunit A proteins from other species. The gene corresponded to a hypothetical C. reinhardtii protein (PNW69996.1). Multiple sequence alignments of the primary structure of this protein with primary structures of RNase H2 subunit A proteins from S. cerevisiae, Arabidopsis thaliana, and Homo sapiens showed patterns of significant conservation (Figure 7). Pairwise similarity between the C. reinhardtii protein and each of the homologous proteins from the three species was statistically significant (E < 1x10-45) when each of the three proteins was used as a query in the Protein Basic Local Alignment Search Tool (BLASTp) search against the C. reinhardtii proteome. Search for genes of RNase H2 subunits B and C was more complicated since these two subunits are less conserved among species related to the alga. In these cases, we had to make a full run of GeneMark-EP+ to re-predict genes in C. reinhardtii genome and, subsequently, to make a second iteration of ProtHint running in a more sensitive mode. As a result, the gene of subunit B was found in positions 1,174,994–1,176,998 in chromosome 7; this gene encoded a protein homologous to 373 out of 719 target RNase H2 subunit B proteins. The homologous gene of subunit C was found in positions 3,889,513–3,890,347 in chromosome 8; this gene encoded a protein homologous to 330 out of 662 target RNase H2 subunit C proteins (Figure 7). As a future direction, it will be interesting to target these potential RNase H2 genes to generate mutants and study how these affect the composition, distribution, and patterns of rNMP presence in the nuclear genome of C. reinhardtii.

Figure 7.

Figure 7

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Hypothetical RNase H2 proteins of C. reinhardtii

Multiple alignment of amino acid sequences of RNase H2 catalytic subunit H2A, and accessory subunits H2B and H2C from H. sapiens, S. cerevisiae, A. thaliana (A. lyrate for subunit C) (Cerritelli and Crouch, 2009) (Kalhorzadeh et al., 2014) and the hypothetical proteins of C. reinhardtii. The C. reinhardtii hypothetical proteins are shown in bold. The highlighted letters show the most conserved parts of the four sequences.

rA found in mitochondrial and chloroplast DNA and rC, rG, and rA found in nuclear DNA are non-uniformly distributed

We then studied whether rA is uniformly distributed in the mitochondrial and chloroplast DNA of the three C. reinhardtii libraries. If, for example, rA is uniformly distributed in C. reinhardtii mitochondrial DNA, the frequency by which the dNMP with base A, C, G, or T is found at position -1 and +1 relative to rA should reflect the frequency of the dinucleotides AA, CA, GA, TA, AC, AG, and AT obtained from the sequence of C. reinhardtii mitochondrial DNA. The heatmap dinucleotide analysis revealed that the frequency of the dNMPs immediately upstream (at position -1) of the rAMP and in part the dNMP at position +1 and -3 from the rAMP in mitochondrial DNA deviates from the expected values but varies among the three mitochondrial libraries (Figures 4B and S4A and Table S3). Possibly, incorporation of rA in mitochondrial DNA may be more sensitive to small variations in growth conditions of the cells used to prepare the ribose-seq libraries analyzed in this study. No bias was found for the nucleotide upstream or downstream of the rAMP at positions -2, +2, +3, -4,+4, -5, +5, -6, +6, -100, and +100 in the mitochondrial DNA (Figure S4). In fact, with the increased gap between dinucleotide pairs (from -2, +2 to -6, +6 and -100 and +100), the uniformity is strengthened, as evidenced by an increased p-value from -1, +1 to -100 and +100 (Table S3). For the chloroplast DNA, the rAMP was preferentially found downstream of dC and more rarely downstream of dA and dT (Figure 5B and Table S3). No particular bias was found for the nucleotide upstream or downstream of the rAMP at positions +1, -2, +2, -3, +3, -4,+4, -5, +5, -6, +6, -100, and +100 in the chloroplast DNA (Figure S4 and Table S3). Due to the low abundance of rC, rG, and rU in the mitochondrial and chloroplast DNA, we did not analyze the surrounding dNMPs of these rNMPs.

For nuclear DNA, we found that both rC and rG are preferentially preceded by dA at position -1. The observed count of NR dinucleotides with an rC or rG (dArC, dCrC, dGrC, and dTrC; dArG, dCrG, dGrG, and dTrG) was significantly different from the expected count calculated for the background frequency of the corresponding dinucleotide pair with the same number of total rCMPs or rGMPs (Table S3). The observed count of the dinucleotide dArC was above the expected count for this pair (Figure 6B and Table S3). Similarly, the observed count of dArG was above the expected count for this pair (Figure 6B and Table S3). For rA, we found that the dArA count was above the expected count for this dinucleotide in all libraries, while for rU, the pattern was less clear, possibly due to the fact that rU is the least abundant rNMP found in the nuclear DNA of C. reinhardtii cells, and more data would be needed to obtain an accurate spectrum of incorporation (Figure 6B and Table S3). A much less prominent difference was found among pair combinations for the dNMPs at position +1 (Figure 6B and Table S3). We also examined the dNMPs at positions -2, +2, -3, +3, -4,+4, -5, +5, -6, +6, -100, and +100. The heatmaps progressively became uniformly darker from -2, +2 to -100, +100, while no particular pattern emerging (Figure S5 and Table S3C). Overall, these results, which were also conserved among all three nuclear libraries, highlight the fact that the dNMPs immediately upstream of the rNMP at position -1 have the most impact on the incorporation of a specific rNMP type in a given genomic position of the algal nuclear DNA. Moreover, these findings demonstrate that rNMPs are not randomly incorporated in the nuclear genome of C. reinhardtii cells.

Discussion

We report a genome-wide analysis of rNMP sites in a photosynthetic organism, the unicellular freshwater green alga C. reinhardtii. We found rNMPs embedded in all genomes of the alga: mitochondrial, chloroplast, and nuclear. We revealed strikingly biased rA incorporation in the mitochondrial and chloroplast DNA but not in the nuclear DNA, in which instead rG and rC are dominant over rA and rU. To our knowledge, there is no previously available report showing that DNA polymerases of C. reinhardtii are prone to incorporate rAMPs in genomic DNA of the alga. C. reinhardtii, like most non-opisthokonts (photosynthetic eukaryotes and protists), does not use DNA polymerase γ in the organelles, like animals and fungi, but the plant and protist organellar DNA polymerase (Moriyama et al., 2011). The disproportionate presence of rA embedded in the mitochondrial and chloroplast DNA reflects the remarkably high ATP/dATP ratio of the cells. These findings support the lack of RNase H2 activity in mitochondrial DNA and provide new evidence for the absence of RNase H2 activity on rNMPs embedded in chloroplast DNA. The inability to cleave and initiate removal of rNMPs embedded in mitochondrial and chloroplast DNA likely allows abundant incorporation of rNMPs in these genomes, particularly rA, significantly and markedly above the frequency expected based on the dA content of the mitochondrial and chloroplast genomes. Work in yeast cells has provided supportive evidence for a frequent exchange of nuclear and mitochondrial dNTP pools (Wanrooij et al., 2017). Thus, although there is no direct proof that this occurs in C. reinhardtii cells, we would expect rA to also be highly incorporated in the nuclear genome of C. reinhardtii. Because rA was not found to be the most frequently incorporated rNMP in the nuclear DNA of C. reinhardtii, but rather rG and rC were more frequently detected, our findings suggest that RNase H2 might have a high workload in removing rA from the nuclear genome of a photosynthetic organism, such as C. reinhardtii. Nevertheless, we were unable to detect an RNase H2 or any other cleavage function from protein extracts of the algal cells to support active removal of rA from nuclear DNA. On the contrary, we were able to find hypothetical C. reinhardtii orthologous genes and proteins for RNase H2A, H2B, and H2C (Figure 7), suggesting that RNase H2 is present and likely functional on rNMPs embedded in C. reinhardtii nuclear DNA. Knowing the position of the hypothetical RNase H2A, H2B, and H2C genes will help to design mutants in these loci to study how defects in RNase H2 genes affect the profile of rNMPs in the nuclear, mitochondrial, and chloroplast genome of the alga. Moreover, it will be valuable to work with a purified RNase H2 enzyme from C. reinhardtii cells to characterize its activity on rNMPs embedded in DNA. There is also the possibility that the nuclear DNA polymerases of C. reinhardtii have a much stronger discrimination capacity for ATP vs. dATP than the DNA polymerases of the mitochondria and chloroplast of the alga. Further studies are needed to understand how rA is specifically excluded from the nuclear DNA of C. reinhardtii cells. In addition, it would also be interesting to investigate the relation between rNMP incorporation in the mitochondrial and chloroplast DNA of the alga and the process of photosynthesis.

If we compare the rNMP content in the mitochondrial DNA of C. reinhardtii with that of S. cerevisiae, S. paradoxus, and S. pombe, we find that rU is the least incorporated rNMP not only in mitochondrial and chloroplast DNA of the alga, as in the yeast mitochondria, but also in nuclear DNA of the alga. Differently, in yeast nuclear DNA, rU is the least frequent rNMP only in RNase H2-defective cells (Balachander et al., 2020). In part, the low level of rU incorporation could reflect the relatively low UTP/dTTP ratio in these eukaryotic cells. Activity of topoisomerase I on sequences with rU (Cho and Jinks-Robertson, 2018; Klein, 2017) and some proofreading activity for nuclear DNA polymerase on rU (Koh et al., 2015) could contribute to the general rare presence of rU in these genomes. Moreover, in each of the three yeast species, rA is incorporated significantly below the expected values (0.25) for the corresponding genomes. Instead, rC and rG are the predominant rNMPs (Balachander et al., 2020). Our results show that the high ATP/dATP ratio in C. reinhardtii cells reflects the composition of rNMPs in the mitochondrial and chloroplast DNA of C. reinhardtii. Interestingly, rA incorporation was found mainly downstream of dC and/or dG in chloroplast DNA. However, no conserved pattern around rA was found in the mitochondrial DNA of all the three ribose-seq libraries of C. reinhardtii. At the same time, rA did display a library-specific preference of incorporation downstream of specific dNMPs in the mitochondrial DNA. It is possible that small variations in growth conditions may affect the incorporation pattern of rA in the algal mitochondrial DNA. While rU is consistently the least represented rNMP in the nuclear, mitochondrial, and chloroplast DNA of C. reinhardtii cells, the mitochondrial DNA also showed very low rG and rC content. Despite highly abundant rA incorporation is a common feature in the mitochondrial and chloroplast DNA of C. reinhardtii, the overall patterns of rNMP incorporation in these two algal organelles are not identical. These results suggest that the two organelles may have different rNMP incorporation mechanisms and/or that the composition of the nucleotide pools in these two organelles is different.

Another conserved feature between yeast and C. reinhardtii rNMP patterns, particularly for the nuclear DNA of the alga, is that the dNMP immediately upstream from the rNMP is the one that has the largest impact on the distribution of rNMPs. In fact, with the increased gap between dinucleotide pairs with an rNMP (from -1, +1 to -100, +100), the uniformity is strengthened not only for nuclear rNMPs but also for the mitochondrial and chloroplast rNMPs, as shown by increased p-value from -1, +1 dinucleotides to −100 and +100 dinucleotides (Tables S3A–S3C). Similarly to what we found for rNMPs in the yeast genome (Balachander et al., 2020), we believe that this biased presence of rNMPs in the algal genome supports an accommodation mechanism by DNA polymerases that facilitates incorporation of rNMPs following specific dNMPs.

In conclusion, via mapping and genome-wide analysis of ribose-seq libraries of the unicellular green alga C. reinhardtii, we have revealed a unique distribution of rNMPs embedded in the DNA of the algal organelles compared to the nuclear DNA of the same cells. It will be interesting to characterize how such disproportionate presence of rA in the genome of the organelles of this photosynthetic organism impacts the DNA metabolic functions of these genomes during their day and night cycles.

Limitations of the study

In this study, we could not prove the activity of the RNase H2 enzyme from C. reinhardtii on rNMP-containing substrates in vitro. Thus, while our data strongly support the presence of a functional RNase H2 enzyme to cleave at rNMPs embedded in the nuclear DNA of C. reinhardtii, we do not provide direct proof of such function. Nonetheless, we have identified the potential genes for the three subunits of RNase H2 in C. reinhardtii. Therefore, we have set the stage for mutation experiments in these genes, to examine the activity of RNase H2 on rNMPs incorporated in the genome of C. reinhardtii cells.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Francesca Storici (storici@gatech.edu).

Materials Availability

All unique/stable reagents generated in this study are available from the Lead contact.

Data and Code Availability

The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files. The Ribose-Map bioinformatics toolkit is available for download at GitHub (https://github.com/agombolay/ribose-map). See also (Gombolay et al., 2019). Custom Python3 scripts for background subtraction is available for download at GitHub under GNU GPL v3.0 license (https://github.com/xph9876/ArtificialRiboseDetection). The custom Python3 scripts for heatmaps is available for download at GitHub under GNU GPL v3.0 license (https://github.com/xph9876/RibosePreferenceAnalysis) Table S2 contains raw data. Bar graphs representing the percentage of each type of rNMP were made using GraphPad Prism 5 (GraphPad Software). The nucleotide sequence context plots were created using custom R scripts. The datasets generated during this study are available at NCBI’s SRA under BioProject ID PRJNA657931. All data generated in this study are available from the Lead contact.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Acknowledgments

We thank the Molecular Evolution Core with A. Bryksin at the Parker H. Petit Institute for Bioengineering and Bioscience of the Georgia Institute of Technology for high throughput sequencing, M.D. Herron and C. Lindsey for supplemental aliquots of C. reinhardtii CC-1690 cells, J. Choi for bioinformatics support, K. Mukherjee and D. Kundnani for critically reading the manuscript, and all members of the Storici laboratory for assistance and feedback on this study. We acknowledge funding from the Egyptian Cultural Affairs & Missions Sector, Cairo, Egypt (to W.M.M.E.), R01GM128145 (to M.B), AI136581 (to B.K.), AI150451 (to B.K.), MH116695 (to. R.F.S), the National Institutes of Health (R01ES026243 to F.S.), and the Howard Hughes Medical Institute (Faculty Scholar grant 55108574 to F.S.) for supporting this work.

Author contributions

Conceptualization, F.S. and W.M.M.E.; Methodology, W.M.M.E., A.L.G., P.X., T.Y., Y.J., S.B., G.N., S.T., N.E.B, T.B., M.B., Y.C., and F.S.; Investigation, W.M.M.E., A.L.G., P.X., T.Y., Y.J., S.B., and F.S.; Writing – Original Draft F.S.; Writing – Review & Editing W.M.M.E., A.L.G., P.X., T.Y., Y.J., S.B., G.N., M.B., B.K., and F.S.; Funding acquisition, M.B., R.F.S., B.K., and F.S.; Resources, M.B., R.F.S., B.K., Y.C., and F.S. All authors commented on and approved the manuscript.

Declaration of interests

We have a patent related to this study: Storici, F., Hesselberth, J.R., and Koh, K. D. Methods to detect ribonucleotides in deoxyribonucleic acids. U.S. Patent U.S. 10,787,703 B1 Sep. 29, 2020.

Published: January 22, 2021

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2020.102005.

Supplemental information

Document S1 Transparent methods, Figures S1–S5, and Table S1
mmc1.pdf (1.6MB, pdf)
Table S2. rNMPs found in mitochondrial, chloroplast and nuclear ribose-seq libraries of C. reinhardtii, related to Figures 2 and 3

List of mitochondrial, chloroplast and nuclear ribose-seq libraries of C. reinhardtii constructed in this study with corresponding data information and indication of algal strain name, genotype, library name, restriction enzyme set used, number of rNMPs, % rA, rC, rG, and rU with mean and standard deviation for each cell compartment, barcode, and number of cycles in PCR 1 and PCR 2.

mmc2.xlsx (20.6KB, xlsx)
Table S3. Comparison of ratios, with P values of heatmap data, related to Figures 4, 5, and 6

Chi-square test is used for (A) mitochondrial, (B) chloroplast, and (C) nuclear DNA to check if the rNMPs are uniformly distributed. The observation value is the count of incorporated rNMP (mono) or dinucleotide with an rNMP (NR or RN, ±1 - ±6, ±100). The expectation is calculated with the corresponding dNMP or dNMP pair background frequency with the same number of total rNMPs. A small p-value (<0.05) means the observation is significantly different with the expectation, which suggests rNMP incorporation is not random. Due to the limit of Excel precision, a p-value less than 10-307 is shown as 0.

mmc3.xlsx (126.2KB, xlsx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1 Transparent methods, Figures S1–S5, and Table S1
mmc1.pdf (1.6MB, pdf)
Table S2. rNMPs found in mitochondrial, chloroplast and nuclear ribose-seq libraries of C. reinhardtii, related to Figures 2 and 3

List of mitochondrial, chloroplast and nuclear ribose-seq libraries of C. reinhardtii constructed in this study with corresponding data information and indication of algal strain name, genotype, library name, restriction enzyme set used, number of rNMPs, % rA, rC, rG, and rU with mean and standard deviation for each cell compartment, barcode, and number of cycles in PCR 1 and PCR 2.

mmc2.xlsx (20.6KB, xlsx)
Table S3. Comparison of ratios, with P values of heatmap data, related to Figures 4, 5, and 6

Chi-square test is used for (A) mitochondrial, (B) chloroplast, and (C) nuclear DNA to check if the rNMPs are uniformly distributed. The observation value is the count of incorporated rNMP (mono) or dinucleotide with an rNMP (NR or RN, ±1 - ±6, ±100). The expectation is calculated with the corresponding dNMP or dNMP pair background frequency with the same number of total rNMPs. A small p-value (<0.05) means the observation is significantly different with the expectation, which suggests rNMP incorporation is not random. Due to the limit of Excel precision, a p-value less than 10-307 is shown as 0.

mmc3.xlsx (126.2KB, xlsx)

Data Availability Statement

The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files. The Ribose-Map bioinformatics toolkit is available for download at GitHub (https://github.com/agombolay/ribose-map). See also (Gombolay et al., 2019). Custom Python3 scripts for background subtraction is available for download at GitHub under GNU GPL v3.0 license (https://github.com/xph9876/ArtificialRiboseDetection). The custom Python3 scripts for heatmaps is available for download at GitHub under GNU GPL v3.0 license (https://github.com/xph9876/RibosePreferenceAnalysis) Table S2 contains raw data. Bar graphs representing the percentage of each type of rNMP were made using GraphPad Prism 5 (GraphPad Software). The nucleotide sequence context plots were created using custom R scripts. The datasets generated during this study are available at NCBI’s SRA under BioProject ID PRJNA657931. All data generated in this study are available from the Lead contact.

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