Friday, July 19, 2013

Strand Asymmetry in Mitochondrial DNA

Funny how the availability of so much free DNA data can go to your head. When I learned that DNA sequence data for more than 2,000 mitochondrial genomes could be accessed, free, at genomevolution.org, I couldn't resist: I wrote some scripts that checked the DNA composition of 2,543 mtDNA (mitochondrial DNA) sequences. What I found blew me away.

If you're a biologist, you're accustomed to thinking of genome G+C (guanine plus cytosine) content as a kind of phylogenetic signature. (Related organisms usually have G+C values that are fairly close to one another.) For purposes of the following discussion, I'm going to reference A+T content, which is, of course, just one-minus-GC. (A GC content of 0.25, or 25%, means the AT content is 0.75, or 75%).

What I learned is that mitochondrial DNA shows strand asymmetry in coding regions (regions that actually get transcribed to RNA, as opposed to non-coding "control" regions and junk DNA). In particular, it shows an excess of pyrimidines (T and C) on the "message strand." This is the exact opposite of the situation in Archaea and bacteria, where message strands tend to accumulate purines (G and A).

The interesting thing is, just like bacteria (and Archaea), mitochondrial genomes tend to show a steady, predictable rate of increase of purines on the message strand with increasing A+T, even though purines are outnumbered by pyrimidines on the message strand. A picture might make this clearer:

Purine (A+G) content versus A+T for the message strand of mitochondrial DNA coding regions (N=2543).

Every point in this graph represents a mitochondrial genome (2,543 in all). As you can see, the regression line (which minimizes the sum of squared error) is upward-sloping, with a rise of 0.149, meaning that for every 10% increase in genome A+T content, there's a corresponding 1.49% increase in message-strand purine (A+G) content. What's striking about this is that in a similar graph for 1,373 bacterial genomes (see this post), the regression-line slope turned out to be 0.148.  Chargaff's second parity law predicts a straight horizontal line at y=0.5. Obviously that law is kaput.

I've written before about my repeated finding (in bacteria, Archaea, eukaryotes, viruses, bacteriophage; basically every place I look) that message-strand purine content accumulates in proportion to genome A+T content. Strand asymmetry with respect to purines and pyrimidines seems to be universal. But why?

Strand-asymmetric buildup of purines or pyrimidines is very hard to explain without invoking either a theory of strand-asymmetric DNA repair or a theory of strand-asymmetric mutagenesis, or both. Is it reasonable to suppose that one strand of DNA is more vulnerable to mutagenesis than another? Yes, if you accept that in a growing cell, the strands spend a good portion of their time apart (during transcription and replication). Neither replication nor transcription is symmetric in implementation. I'll spare you the details for the replication side of the argument, but suffice it to say, replication-related asymmetries are not likely (in my opinion) to be behind the purine/pyrminidine strand asymmetries I've been documenting. What we're seeing, I think, is the result of asymmetric repair at transcription time.

During transcription, a gene's DNA strands are separated. One strand is used as a template by RNA polymerase to create messenger RNA and ribosomal RNA. The other strand is free and floppy and vulnerable to attack by mutagens. But it's also readily accessible to repair enzymes.


The above diagram oversimplifies things considerably, but I include it for the benefit of non-biogeeks who might want to follow this argument through. Note that DNA strands have directionality: the sugar bonds face one way in one strand and the other way in the other strand. This is denoted by the so-called 5'-to'3 orientation of strands.(RNAP = RNA polymerase.)

DNA repair is a complex subject. Be assured, every cell, of every kind, has dozens of different kinds of enzymes devoted to DNA repair. Without these enzymes, life as we know it would end, because DNA is constantly undergoing attack and requiring repair.

The Ogg family of DNA base-excision enzymes exhibit
a signature helix-hairpin-helix topology (HhH). See
Faucher et al., Int J Mol Sci 2012; 13(6): 6711–6729.
Some types of repair take place in double-stranded DNA (that is, DNA that is not undergoing replication or transcription). Other types of repair apply to single-stranded DNA. In bacteria as well as higher life forms, there's a transcription-coupled repair system (TCRS) that comes into play when RNA polymerase is stalled by thymine dimers or other DNA damage. This remarkably elaborate system changes out short sections of damaged DNA (at considerable energy cost). Because it involves replacing whole nucleotides (sugar and all), it's categorized as a Nucleotide Execision Repair system (NER). The alternative to NER is Base Excision Repair (BER), which is where a defective base (usually an oxidized guanine) gets snipped out without removing any sugars from the DNA backbone. The enzymes that perform this base-clipping are generically known as glycosylases.

For many years, it was thought that mitochondria did not have DNA repair systems. We now know that's not true. Mitochondrial DNA is subject to constant oxidative attack and it turns out the damage is quickly repaired, in double-stranded DNA. Evidence for repair of single-stranded mtDNA is scant. Those who have looked for a transcription-coupled repair system (or indeed any NER system) in mitochondria have not found one. Mitochondrial BER repair (via Ogg1) does exist, but it seems to operate when the DNA is double-stranded, not during transcription. This makes sense, because for BER to finish, the strand must be nicked by AP endonuclease after the bad base is popped out, then the repair proceeds by matching the opposing base (opposite the abasic site) using the other strand as template. In Clostridia and Archaea (which have an Ogg enzyme that other bacteria do not have; see this post and this paper), Ogg1 can pop out a bad base while the DNA is single-stranded; Ogg1 then binds to the abasic site and is only released by AP endonuclease when it arrives later on.

Bottom line, we know that mitochondrial DNA spends much of its time in the unwound state (because mtDNA products are very highly transcribed) and that the non-transcribed DNA strand is extremely vulnerable to oxidative attack. (The template strand is less vulnerable, because it is cloaked in enzymes: RNA polymerase, transcription factors, ribosomes, etc.) We also know that 8-oxoguanine is the most prevalent form of oxidative damage in mtDNA and that, uncorrected, such damage leads to G-to-T transversion. The finding of consistently high pyrimidine content in the message strand of mitochondrial DNA (see graph further above) is consistent with a slower rate of repair of the non-transcribed strand, and the differential occurrence of G-to-T transversions on that strand. Or at least, that's a possible explanation of the pyrimidine richness of the message strand of mtDNA.

But there are additional factors to consider, such as selection pressure. Mitochondrial DNA tends to encode membrane-associated proteins, and membrane proteins use nonpolar amino acids, which are (in turn) predominantly encoded by pyrimidine-rich codons. More about this in an upcoming post.

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