Kinetoplastid DNA

It seems to be that time of the year in which we post about something to do with our posts, as these are the topics we are supposed to be reading about now. Therefore, a post on parasites. That’s where the connection with my project finishes, but anyway.

As you might know, tryponosomatids are organisms from the family Troponosomatidae, which include some pretty nasty unicellular parasites such as Trypanosoma brucei, causer for African sleeping disease, and Leishmania, causer of leishmaniasis. As other higher eukaryotes, these parasites are characterised by two genomes: a nuclear genome and a mitochondrion genome, 10-30% of all the cell DNA, situated in the kinetoplast (the name given to the mitochondrion in these organisms).

Fig.1 A- Schematic minicircle organization. B- in vivo network organization, seen sideways.

(Adapted from reference 1)

So far nothing extraordinary. Things get interesting, when one looks at the structure of the kinetoplast DNA (kDNA) in these species. The kDNA is constituted by thousands of small DNA circles, the minicircles, and a few dozen of large DNA circles, the maxicircles. These are interlocked, as in a chain mail of medieval armour. Each circle is interlocked with 3 other neighbouring circles. The minicircles and maxicircles are stretched into a disk-shaped structure, situated near to the flagellar basal body (Fig.1). The maxicircles are those more similar to normal mitochondrion genome. They include rRNAs and genes encoding proteins associated with the respiratory processes that takes place in this organelle. However, the maxicircles mRNA requires extensive editing, namely the introduction or deletion of uridylate. The minicircles encode the guide RNAs used as templates for this editing. Since there are many types of editing required, as well as maxicircles with different sequences, there is the need for the thousand minicircles with different sequences. Seems a bit of a wasteful system, but what do I know!

More interesting is to think of the replication process. The minicircles must stop being interlocked, replicated, interlocked again, and in the end originate two new networks of thousands of minicircles that separate into the two new daughter cells. Let us look at the different steps, following an individual minicircle (Fig.2):

Fig.2 kDNA replication model. (From reference 2)

1- A minicircle must first be released from the network into the so-called kinetoflagellar zone (KTZ), where several proteins involved in the process exist. Here the unidirectional replication of the circle occurs, although the details of this process are not well known.

2- The two daughter minicircles move to opposite antipodal sites of the circle, where there are two protein assemblies. Here a variety of reactions occur: the RNA primers are removed, the gaps between Okazaki fragments are filled by DNA pol, and nick as sealed by DNA ligase. An important characteristic, however, is that the minicircles maintain at least one gap in their structure. This is thought to be a self-check feature of this system, as a way to guarantee that no minicircle is replicated twice.

3- Minicircles are attached by topoisomerase II to the network periphery adjacent to the antipodal sites.

The way the minicircles are attached to the disk varies with the species of trypanosomatid. In T.brucei, for example, the minicircles accumulate at the network poles. In T.cruzi, and C.fasciculata, the minicircles seem to be uniformly attached around the periphery of the disk, creating a ring of new minicircles, of increased thickness. How this uniform attachment is done is not certain, but it is though that either the antipodal protein complexes or the disk itself must rotate! (Fig.3)

Fig.3 Replication in C.fasciculata. New minicircles have been labelled with fluorescent nucleotides. The arrangement of the new minicircles around the periphery of the disk is obvious (from reference 2).

4- Regardless of the method, new minicircles are attached to the disk, and the valence must increase to 4 to 6 attachments per minicircle, in order to ensure twice as many minicircles in the same space (the mitochondrion membrane has not doubled at this point). When the space increases, topoisomerase II ensures that valence returns to 3.

5- Finally, the nicks are repaired and the network splits into 2, although how exactly this division occurs is not well known. Most importantly, it is not known for sure how to guarantee that the two daughter networks have the exactly same minicircles. Considering their importance in maxicircle mRNA editing, the loss of one minicircle could have dramatic consequences.

There is much to be explained in this process, namely identify exactly how the minicircles are moved to the different antipodal sites, or the details of individual minicircle replication, but such understanding will require the identification of the proteins involved. Considering the complexity of the process, many must be involved, but only a few have been identified so far.

This is probably not life-changing research, but it is surely interesting. It is important to remind ourselves once in a while that though we learn the textbook pathways and processes, Nature seems to like using hundreds of alternatives. Just to make scientists lives harder, I assume!

Liu, B., Liu, Y., Motyka, S., Agbo, E., & Englund, P. (2005). Fellowship of the rings: the replication of kinetoplast DNA Trends in Parasitology, 21 (8), 363-369 DOI: 10.1016/j.pt.2005.06.008

(2) Smith D. and Parsons M. (1996). Molecular Biology of Parasitic Protozoa, Kinetoplast DNA: Structure and Replication, Oxford University Press, USA