The role of telomeres in poxvirus DNA replication

The poxvirus telomere consists of at least three DNA domains, the AT-rich incompletely base-paired hairpin terminus, the nearby region of repeats, and the intervening TRT domain. The only sequence to which a definite function has been ascribed to date is the TRT domain, which constitutes the target region necessary for telomere resolution. The strict conservation of AT-richness and incomplete base-pairing of the hairpin termini strongly imply their essential function in the life cycle of poxviruses. Similarly, the identical location of the region of repeats, irrespective of their complexity, implies that these elements fulfill some essential function(s). Yet, neither the hairpin nor the region of repeats is obligatorily required in the process that converts the replicative intermediate configuration into hairpin termini. Possible functions that can be ascribed to either or both of these DNA elements are: (a) initiation of DNA replication; (b) packaging of the genome into the virion core; (c) an attachment site to promote telomere-matrix or telomere-telomere association; (d) isomerization of adjoining hairpin termini to generate the replicative intermediate telomere fusion configuration; (e) transient maintenance of the dimer junction configuration prior to resolution; and (f) recombination between genomic termini, which may be an essential step during poxvirus DNA replication. The challenge for the future will be to develop new and innovative approaches to define the function(s) of these telomere regions. The development of the transfection assay has been one such approach which, in a relatively short time, has contributed greatly to our current level of understanding of telomere resolution. To further adapt this assay to study other potential functions, such as initiation of DNA replication, it is necessary to gain a better understanding of the mechanism of the apparently origin-independent replication of transfected plasmid DNA. Such studies may well answer the question whether poxviruses in fact use defined origins of replication. The efficient replication and resolution of plasmid DNA in poxvirus-infected cells has allowed determination of the domain boundaries of a telomeric region (TRT) that is required in cis for the resolution of the telomere fusion element to hairpin termini. This region was shown to contain a minimal core region, which is absolutely required for resolution, and an auxiliary region which promotes optimal resolution efficiency. The availability of cloned wild type and mutant substrates will, in all likelihood, lead to an efficient in vitro resolution system. The development of such a system is now within reach and should, in combination with genetic studies, allow identification of the various components necessary to generate hairpin termini from replicative intermediate telomere fusion elements. Finally, a better understanding of molecular mechanisms utilized for poxviral telomere replication and resolution will help to resolve the issue of whether or not common pathways or analogous mechanisms are also utilized for higher order eukaryotic chromosomes.