Chargaff's Second Parity Rule is Violated in Proportion to Genome A+T Content

Erwin Chargaff was the first to notice, in the early 1950s, before Watson and Crick deduced the structure of DNA, that the quantity of purines in DNA equals the quantity of pyrimidines (specifically, the amount of adenine equals the amount of thymine; and the amount of guanine equals the amount of cytosine). This observation was key to establishing the structure of DNA, and it is often cited as Chargaff's first parity rule. But Chargaff also made another observation (the second parity rule), namely that even within a single strand of DNA, the amount of adenine tends to equal the amount of thymine and the amount of guanine tends to equal the amount of cytosine.

It's easy to understand why the first parity rule holds true, because complementarity of DNA strands depends on A pairing with T and G pairing with C; these pairings give rise to the "rungs" of the DNA ladder and ensure that copying of strands occurs with total fidelity during cell division. But there doesn't seem to be any a priori reason why the second parity rule should hold true. And in fact, it often doesn't hold true, as Wacław Szybalski noted in 1966 when he reported finding imbalances of purines and pyrimidines in bacteriophage and other DNA samples. Szybalski observed that in most cases, protein-coding regions of DNA tend to have slightly more purines than pyrimidines on one strand and slightly more pyrimidines than purines on the other strand, such that messenger RNA ends up purine-heavy.

If you're having trouble visualizing the situation, imagine a very short (12-base) "chromosome" containing 50% G+C content. One possibility is that one strand looks like GGGGGGTTTTTT and the other strand is CCCCCCAAAAAA. In this case half the purines (all the G's) are on one strand and half (A's) are on the other. But you could just as easily have strands be GGGGGGAAAAAA and CCCCCCTTTTTT. In this case, one strand is all-purines, the other all-pyrimidines. Both examples violate Chargaff's second rule, which requires that G = C and A = T within each strand (e.g., GGGCCCTTTAAA + CCCGGGAAATTT would obey the rule).

To my knowledge, no one has yet reported the fact (which I'll now report) that the degree to which Chargaff's second parity rule is violated depends on the G+C content of the source genome (at least for bacteria). Simply put, organisms with a G+C content of around 68% obey Chargaff's rules. Organisms with more than 68% G+C content violate Chargaff's second rule in the direction of pyrimidine loading of mRNA. Organisms with less than 68% G+C content (which of course includes the overwhelming majority of organisms) have purine-heavy DNA, to a degree that depends on the amount of A+T in the DNA.

Purine/pyrimidine ratio (in coding regions) as a function of genome G+C content based on codon analysis of 93 organisms. As genomes become more A+T rich, mRNA becomes more heavily purine-loaded.

The above graph shows how this relationship works. To create the graph, I did a statistical analysis of codon usage in 93 bacterial species. Organisms were chosen so as to obtain representatives across the AT/GC spectrum. No genus is represented more than once. In order to get as broad a sampling as possible, I included 14 intracellular symbionts with ultra-low G+C content (plus one such creature—Candidatus Hodgkinia cicadicola—with a 58% G+C content); many extremophiles; heterotrophs and autotrophs; pathogens and non-pathogens; and organisms with large and small genomes. The complete organism list is presented in a table further below.

Codon usage statistics for each organism were obtained using tools at http://genomevolution.org. Relative prevalences of A, T, G, and C in the genomes' coding regions were determined by codon frequency analysis. The purine:pyrimidine ratio was simply calculated as (A+G)/(C+T) based on the codon-wise frequency of usage of each base.

What we see is that while there is a good deal of noise in the data, nevertheless it's quite clear that purine/pyrimidine ratios increase sharply as genome G+C decreases.Organisms for which Chargaff's second rule holds true (points falling at y = 1.0) are in a small minority. Most organisms have purine-rich coding regions, resulting in purine-rich mRNA.

Purine enrichment occurs for both adenine and guanine. For example, in Clostridium botulinum (genome G+C = 28.21%), codon analysis reveals G/C/A/T relative abundances (on the coding strand) of 18.3/10.8/40.3/30.6.

Intra-codon base position analysis reveals that purine enrichment is far more concentrated in position one of the codon than other positions. The graphs below show the purine balance on a position-by-position basis, for each base in a codon.

Most of the variation in purine/pyrimidine ratio happens in position 1 of the codon (the 'A' in ATG, for example). Notice that the purine/pyrimidine ratio in this position is well above 1.0 for all organisms.

Variation in purine loading at the second position of the codon is more carefully controlled (notice that there is less "scatter" in this graph). The y-axis scale is different here than in the previous graph, hence the slope is quite a bit less pronounced than it looks. Also, notice that most of the points in this plot are below parity (i.e., below 1.0 on the y-axis), indicating that this codon position is relatively pyrimidine-rich.

The third (so-called "wobble") position of the codon shows considerable variation in values, but the slope of the curve is less than in the previous two graphs, and this position is pyrimidine-rich for about two-thirds of the organisms.

It's well known that GC-skew tends to be exaggerated in position 3 of the codon. For example, if the overall genome G+C is 70%, the position-wise G+C for the wobble base may be 90%. Surprisingly, we find that purine loading is most exaggerated in position 1 of the codon, not position 3. Not only is the slope of the purine-ratio curve shallower in position 3 than for the other two base positions, only position 1 is actually purine-heavy: positions 2 and 3 tend to be net pyrimidine-rich. This fact (that purine loading is primarily localized to codon position 1, whereas GC-skew is exaggerated in position 3) might indicate that the forces responsible for purine loading are entirely different from the forces responsible for GC skew.

What might those forces be? What kinds of selection pressure might cause organisms to purine-load one strand of their DNA? One possibility is that purine loading of the coding strand is a strategy for protecting the "weaker" or more vulnerable strand from damage or mutations. Cytosine is thought to be particularly vulnerable to deamination (and later substitution with thymine, during repair). It's possible that the transcription process (which is asymmetric, in that RNA polymerase operates against just one strand of DNA, leaving the other strand free) is protective of the antisense strand of DNA. That is, in transcription, RNA polymerase cloaks the antisense strand and in so doing renders that strand less vulnerable to deamination events, rogue methylations, etc., while transcription is taking place.

An entirely different possibility is envisioned by an RNA World hypothesis. In this hypothesis, the genetic material of early ancestor organisms was single-stranded RNA. Since single-stranded RNA is not "complementary" to anything, there is no need for it to obey Chargaff symmetries. Thus, purine loading could have occurred prior to the advent of double-stranded DNA, and early organisms could have been uniformly AT-rich. In this model of the world, GC-rich genomes are a late development, and the processes responsible for creating GC-rich DNA led to genetic material with full Chargaff base parity.

We may not know for a long time (if ever) what the mechanisms of purine enrichment are. But we know for sure that purine accumulation is a widespread phenomenon in the bacterial world (operating across diverse clades) and happens in a way that encourages purine-rich mRNA in organisms with low G+C content in their genomes.


Organisms used in this study:

Organism	GC%	genome size
Anaeromyxobacter dehalogenans 2CP-1	74.67	5009007
Cellulomonas flavigena strain DSM 20109	74.29	4123179
Xylanimonas cellulosilytica strain DSM 15894	72.47	3831380
Streptomyces bingchenggensis strain BCW-1	70.75	11936683
Myxococcus fulvus strain HW-1	70.63	9003593
Rubrobacter xylanophilus strain DSM 9941	70.48	3225748
Rhodospirillum centenum ATCC 51521	70.46	4355543
Actinomyces sp. oral taxon 175 strain F0384	68.73	3133330
Rhodococcus equi strain ATCC 33707	68.72	5259057
Acidovorax avenae subsp. citrulli strain AAC00-1	68.53	5352772
Bordetella bronchiseptica strain RB50	68.08	5339179
Alicycliphilus denitrificans strain K601	67.81	5070751
Stenotrophomonas maltophilia strain JV3	66.89	4544477
Rhodobacter capsulatus strain SB 1003	66.56	3871920
Pseudomonas aeruginosa strain PA7	66.45	6588339
Ralstonia eutropha strain H16	66.29	7416678
Xanthomonas campestris pv. raphani strain 756C	65.29	4941214
Thioalkalivibrio sp. strain HL-EbGR7	65.06	3470516
Rhodopseudomonas palustris strain BisB18	64.96	5513844
Brevundimonas diminuta strain ATCC 11568	64.51	3369316
Rhodothermus marinus strain DSM 4252	64.09	3386737
Bradyrhizobium japonicum strain USDA 110	64.06	9105828
Mycobacterium tuberculosis strain C	63.82	4379118
Thermanaerovibrio acidaminovorans strain DSM 6589	63.79	1848474
Halomonas elongata DSM 2581 strain type DSM 2581	63.61	4061296
Novosphingobium nitrogenifigens strain DSM 19370	63.43	4182647
Polaromonas sp. strain JS666	62.24	5898676
Desulfovibrio africanus strain Walvis Bay	61.42	4200534
Candidatus Desulforudis audaxviator strain MP104C	60.85	2349476
Burkholderia rhizoxinica strain HKI 454	60.68	3750138
Slackia heliotrinireducens strain DSM 20476	60.21	3165038
Candidatus Nitrospira defluvii	59.03	4317083
Halogeometricum borinquense DSM 11551	58.43	3944467
Candidatus Hodgkinia cicadicola strain Dsem	58.39	143795
Sideroxydans lithotrophicus strain ES-1	57.54	3003656
Cenarchaeum symbiosum A	57.37	2045086
Serratia sp. strain AS12	55.96	5443009
Acidaminococcus fermentans strain DSM 20731	55.84	2329769
Hyperthermus butylicus strain DSM 5456	53.74	1667163
Methanosaeta thermophila (Methanothrix thermophila PT) strain PT	53.55	1879471
Neisseria gonorrhoeae strain NCCP11945	53.37	2236178
Treponema paraluiscuniculi strain Cuniculi A	52.74	1133390
Pseudovibrio sp. strain FO-BEG1	52.38	5916782
Nitrosococcus halophilus strain Nc4	51.60	4145260
Herpetosiphon aurantiacus DSM 785	50.84	6785430
Escherichia coli B strain REL606	50.77	4629812
Bdellovibrio bacteriovorus strain ATCC15356;	50.65	3782950
Pectobacterium wasabiae strain WPP163	50.48	5063892
Anaplasma centrale (Anaplasma marginale subsp. centrale str. Israel) strain Israel	49.98	1206806
Actinomyces coleocanis strain DSM 15436	49.47	1723843
Desulfotalea psychrophila strain LSv54	46.72	3659634
Polynucleobacter necessarius strain STIR1	45.56	1560469
Nitrosomonas sp. strain Is79A3	45.44	3783444
Coprothermobacter proteolyticus strain DSM 5265	44.77	1424912
Vibrio sp. Ex25 strain EX25	44.57	5160431
Geobacillus thermoglucosidans strain TNO-09.020	43.82	3740238
Waddlia chondrophila strain 2032/99	43.59	2139757
Bacteroides fragilis strain 638R	43.42	5373121
Thiomicrospira crunogena strain XCL-2	43.13	2427734
Coxiella burnetii strain CbuG_Q212	42.63	2008870
Chlamydia muridarum Nigg strain MoPn	40.27	1080451
Psychromonas ingrahamii strain 37	40.09	4559598
Nitratiruptor sp. strain SB155-2	39.69	1877931
Lactobacillus reuteri strain DSM 20016	38.87	1999618
Thermotoga lettingae strain TM	38.70	2135342
Streptococcus pyogenes strain Alab49	38.63	1841271
Bartonella bacilliformis strain ATCC 35685; KC583	38.24	1445021
Halothermothrix orenii strain DSM 9562; H 168	37.78	2463968
Staphylothermus marinus strain F1	35.73	1570485
Calditerrivibrio nitroreducens strain DSM 19672	35.69	2216552
Bacillus thuringiensis serovar andalousiensis strain BGSC 4AW1	34.96	5488844
Desulfurobacterium thermolithotrophum	34.95	1541968
Wolbachia pipientis strain wPip	34.19	1482455
Nitrosopumilus maritimus strain SCM1	34.17	1645259
Staphylococcus aureus strain 04-02981	32.90	2821452
Methanobrevibacter ruminantium strain M1	32.64	2937203
Rickettsia japonica strain YH	32.35	1283087
Methanocaldococcus fervens strain AG86 (v1)	32.21	1507251
Mycoplasma genitalium G37 strain G-37	31.69	580076
Nanoarchaeum equitans strain Kin4-M	31.56	490885
Orientia tsutsugamushi strain Boryong	30.53	2127051
Methanococcus aeolicus strain Nankai-3	30.04	1569500
Candidatus Pelagibacter ubique strain HTCC1062	29.68	1308759
Ehrlichia canis strain Jake	28.96	1315030
Arcobacter nitrofigilis strain DSM 7299	28.36	3192235
Clostridium botulinum A strain ATCC 19397	28.21	3863450
Parvimonas sp. oral taxon 393 strain F0440	28.17	1483165
Candidatus Arthromitus sp. strain SFB-mouse-NYU	27.94	1569870
Candidatus Blochmannia floridanus	27.38	705557
Buchnera aphidicola (Acyrthosiphon pisum) strain 5A	25.69	653223
Wigglesworthia glossinidia endosymbiont of Glossina brevipalpis	22.48	703004
Candidatus Sulcia muelleri strain CARI (v1)	21.13	276511
Candidatus Carsonella ruddii strain PV (v1)	16.56	159662