Recombination, Mutagenesis and Evolution of Phage T4 DNA

对迅速扩大的序列数据库的比较表明，在进化规模上存在广泛的基因横向交换和转移。转座元件，包括逆转录病毒，以及病毒和质粒的位点特异性重组系统，已被认为与侧向基因转移有关。最近的结果表明，侧向基因转移也可以使用同源重组机制，在同源性有限的序列之间启动，并且多个突变可以或多或少地同时出现在相邻区域。这表明，DNA序列的横向转移可以同时导致新基因的获得、多部分控制元件的进化，如DNA复制的起源，以及相当大的序列分歧。为了解释这些观察，提出了一个模型，在该模型中，在部分匹配的序列之间形成异源双链。随后的异源双链修复同时产生多个突变，依赖于重组的DNA复制分解重组中间产物，并将一些未改变的外源DNA拼接到常驻基因组中。强大的选择压力维持了那些编码功能必需基因的重组体。该模型暗示，类似的重组和修复步骤在横向基因转移的“幸存者”之间产生了序列差异，现在又在他们之间产生了重组障碍。这个项目的主要目的是测试这个模型，目的是更好地理解重组过程及其对基因突变和进化的影响。在这些研究中，噬菌体T4和相关噬菌体(T-evens)有几个优势：1)高重组潜力，2)包装的DNA末端和造成的断裂对重组的刺激，3)对重组和DNA复制的明显冗余、交织和非线性途径的理解，4)认识到这些途径的酶在不同的复合体中以不同的组合混合和匹配，执行不同的功能，从而对不同的重组途径和DNA复制的启动产生不同的影响。在上述假设的框架内，结合遗传学、基因组学和生化方法，以下问题正在被解决：1)通过横向基因转移获得的基因内部或附近产生的明显序列差异有多大？2)序列差异在多大程度上导致相关噬菌体对某些噬菌体的排斥(例如，T4不包括T2，RB69不包括T4)？3)哪些噬菌体或宿主编码的复制、重组、修复蛋白和限制性内切酶参与了这种排除？以及4)人们可以在多大程度上解释来自不同生物的同源和同源蛋白质的碱基序列比氨基酸序列更大的差异，这是同源重组导致的侧向转移的突变潜力的结果？主要的策略是使用包含来自不同T-甚至这些基因的噬菌体和嵌合体的同源但不同的基因的噬菌体和质粒。噬菌体和宿主重组以及限制性内切酶对重组的影响将在后代的生存能力以及潜在的异源双链的形成、持续或消除上进行测试。后者将通过非变性DNA的Southern blotting和包装的后代DNA的测序进行监测。了解这些重组和排斥机制对于解释分支、系统发育树和进化节奏是相关的。这对于理解产生抗体多样性的“错误”也是相关的。

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.