Recombination, Mutagenesis and Evolution of Phage T4 DNA

Vanderbilt University, Nashville TN

Investigators

Abstract

Comparisons of rapidly expanding sequence databases suggest that there has been extensive lateral exchange and transfer of genes on an evolutionary scale. Transposable elements, including retroviruses, and site specific recombination systems of viruses and plasmids have been implicated in lateral gene transfer. Recent results have indicated that lateral gene transfer can also use homologous recombination mechanisms, initiated between sequences of limited homology, and that multiple mutations can appear more or less simultaneously in an adjacent region. This suggests that lateral transfer of DNA sequences can simultaneously lead to acquisition of new genes, the evolution of multi-partite control elements, such as origins of DNA replication, and to considerable sequence divergence. To explain these observations, a model is proposed in which heteroduplexes are formed between partially matching sequences. Subsequent heteroduplex repair simultaneously generates multiple mutations, and recombination-dependent DNA replication resolves the recombination intermediates and splices some unchanged foreign DNA into the resident genome. Strong selection pressure maintains those recombinants that code for functional essential genes. The model implies that similar recombination and repair steps that generated the sequence divergence between the 'survivors' of lateral gene transfer now generate recombinational barriers between them. The major aim of this project is a test of this model, with the goal of better understanding recombination processes and their consequences for mutagenesis and evolution of genes. Phage T4 and related phages (T-evens) have several advantages for these studies: 1) a high recombination potential, 2) the demonstrated stimulation of recombination by packaged DNA ends and inflicted breaks, 3) an understanding of apparently redundant, interwoven and non-linear pathways of recombination and DNA replication and 4) an appreciation that the enzymes of these pathways are mixed and matched in various combinations in different complexes, performing different functions that differentially affect different pathways of recombination and of initiation of DNA replication. Within the framework of the hypotheses outlined above, the following questions are being addressed, combining genetic, genomic and biochemical approaches: 1) to what extent are apparent sequence differences generated within or in the vicinity of genes that had been acquired by lateral gene transfer? 2) to what extent are sequence differences responsible for exclusion of certain phages by related phages (e. g., T4 excludes T2, and RB69 excludes T4 )? 3) which phage- or host-encoded replication, recombination, and repair proteins and restriction enzymes participate in such exclusion? and 4) to what extent can one explain the larger differences in base sequences of genes than in amino acid sequences of orthologous and paralogous proteins from different organisms as consequences of the mutagenic potential of lateral transfer by homologous recombination? The main strategy is to use phages and plasmids containing homologous, but diverged genes from different T-even phages and chimeras of these genes. Effects of phage and host recombination and restriction enzymes on recombination will be tested on viability of the progeny and on formation, persistence or elimination of potential heteroduplex loops. The latter will be monitored by Southern blotting of non-denatured DNA and by sequencing of packaged progeny DNA. Understanding these recombination and exclusion mechanisms is relevant to interpretations of clades, phylogenetic trees and tempos of evolution. It is also relevant for understanding 'errors' that can generate antibody diversity.