Structural and Functional Characteristics of Homing Endonucleases

Mobile genetic elements constitute a remarkably diverse group of nonessential selfish genes that provide no apparent function to the host. These selfish genes have been implicated in host extinction, speciation and architecture of genetic systems. Homing endonucleases, encoded by the open reading frames embedded in introns or inteins of mobile genetic elements, possess double-stranded DNA-specific endonuclease activity. They inflict sequence-specific double-strand breaks at or near the homing site in intron- or intein-less allele. Subsequently, through nonreciprocal exchange the insertion sequence (intron or intein) is transferred from an intein- or intron-containing allele to an intein- or intron-less allele. The components of host double-strand break repair pathway are thought to finish the “homing” process. Several lines of evidence suggest that homing endonucleases are capable of promoting transposition into ectopic sites within or across genomes for their survival as well as dispersal in natural populations. The occurrence of inteins at high frequencies serves as instructive models for understanding the mechanistic aspects of the process of homing and its evolution. This review focuses on genetic, biochemical, structural, and phylogenetic aspects of homing endonucleases, and their comparison with restriction endonucleases.     

The evolution of homing endonuclease genes and group I introns in nuclear rDNA

Group I introns are autonomous genetic elements that can catalyze their own excision from pre-RNA. Understanding how group I introns move in nuclear ribosomal (r)DNA remains an important question in evolutionary biology. Two models are invoked to explain group I intron movement. The first is termed homing and results from the action of an intron-encoded homing endonuclease that recognizes and cleaves an intronless allele at or near the intron insertion site. Alternatively, introns can be inserted into RNA through reverse splicing. Here, we present the sequences of two large group I introns from fungal nuclear rDNA, which both encode putative full-length homing endonuclease genes (HEGs). Five remnant HEGs in different fungal species are also reported. This brings the total number of known nuclear HEGs from 15 to 22. We determined the phylogeny of all known nuclear HEGs and their associated introns. We found evidence for intron-independent HEG invasion into both homologous and heterologous introns in often distantly related lineages, as well as the "switching" of HEGs between different intron peripheral loops and between sense and antisense strands of intron DNA. These results suggest that nuclear HEGs are frequently mobilized. HEG invasion appears, however, to be limited to existing introns in the same or neighboring sites. To study the intron-HEG relationship in more detail, the S943 group I intron in fungal small-subunit rDNA was used as a model system. The S943 HEG is shown to be widely distributed as functional, inactivated, or remnant ORFs in S943 introns.     

Domain loss has independently occurred multiple times in plant terpene synthase evolution

The extensive family of plant terpene synthases (TPSs) generally has a bi-domain structure, yet phylogenetic analyses consistently indicate that these synthases have evolved from larger diterpene synthases. In particular, that duplication of the diterpene synthase genes required for gibberellin phytohormone biosynthesis provided an early predecessor, whose loss of a approximately 220 amino acid 'internal sequence element' (now recognized as the γ domain) gave rise to the precursor of the modern mono- and sesqui-TPSs found in all higher plants. Intriguingly, TPSs are conserved by taxonomic relationships rather than function. This relationship demonstrates that such functional radiation has occurred both repeatedly and relatively recently, yet phylogenetic analyses assume that the 'internal/γ' domain loss represents a single evolutionary event. Here we provide evidence that such a loss was not a singular event, but rather has occurred multiple times. Specifically, we provide an example of a bi-domain diterpene synthase from Salvia miltiorrhiza, along with a sesquiterpene synthase from Triticum aestivum (wheat) that is not only closely related to diterpene synthases, but retains the ent-kaurene synthase activity relevant to the ancestral gibberellin metabolic function. Indeed, while the wheat sesquiterpene synthase clearly no longer contains the 'internal/γ' domain, it is closely related to rice diterpene synthase genes that retain the ancestral tri-domain structure. Thus, these findings provide examples of key evolutionary intermediates that underlie the bi-domain structure observed in the expansive plant TPS gene family, as well as indicating that 'internal/γ' domain loss has occurred independently multiple times, highlighting the complex evolutionary history of this important enzymatic family.     

Reductions in complexity of mitochondrial genomes in lichen‐forming fungi shed light on genome architecture of obligate symbioses

Symbioses among co‐evolving taxa are often marked by genome reductions such as a loss of protein‐coding genes in at least one of the partners as a means of reducing redundancy or intergenomic conflict. To explore this phenomenon in an iconic yet under‐studied group of obligate symbiotic organisms, mitochondrial genomes of 22 newly sequenced and annotated species of lichenized fungi were compared to 167 mitochondrial genomes of nonlichenized fungi. Our results demonstrate the first broad‐scale loss of atp9 from mitochondria of lichenized fungi. Despite key functions in mitochondrial energy production, we show that atp9 has been independently lost in three different lineages spanning 10 of the 22 studied species. A search for predicted, functional copies of atp9 among genomes of other symbionts involved in each lichen revealed the full‐length, presumably functional copies of atp9 in either the photosynthetic algal partner or in other symbiotic fungi in all 10 instances. Together, these data yield evidence of an obligate symbiotic relationship in which core genomic processes have been streamlined, likely due to co‐evolution.     

Invasion genetics of the Mediterranean fruit fly: variation in multiple nuclear introns

Biological invasions generally start from low initial population sizes, leading to reduced genetic variation in nuclear and especially mitochondrial DNA. Consequently, genetic approaches for the study of invasion history and population structure are difficult. An extreme example is the Mediterranean fruit fly, Ceratitis capitata (Medfly), for which successive invasions during this century have resulted in a loss of 60% of ancestral genetic variation in isozymes and 75% of variation in mitochondrial DNA. Using Medflies as an example, we present a new approach to invasion genetics that measures DNA sequence variation within introns from multiple nuclear loci. These loci are so variable that even relatively recently founded Medfly populations within California and Hawaii retain ample genetic diversity. Invading populations have only lost 35% of the ancestral genetic variation. Intron variation will allow high-resolution genetic characterization of invading populations in both natural and managed systems, although non-equilibrium methods of analysis may be necessary if the genetic diversity represents sorting ancestral polymorphism.