Abstract
The recombination activity of Escherichia coli (E. coli) RecA protein reflects an evolutionary balance between the positive and potentially deleterious effects of recombination. We have perturbed that balance, generating RecA variants exhibiting improved recombination functionality via random mutagenesis followed by directed evolution for enhanced function in conjugation. A recA gene segment encoding a 59 residue segment of the protein (Val79-Ala137), encompassing an extensive subunit-subunit interface region, was subjected to degenerate oligonucleotide-mediated mutagenesis. An iterative selection process generated at least 18 recA gene variants capable of producing a higher yield of transconjugants. Three of the variant proteins, RecA I102L, RecA V79L and RecA E86G/C90G were characterized based on their prominence. Relative to wild type RecA, the selected RecA variants exhibited faster rates of ATP hydrolysis, more rapid displacement of SSB, decreased inhibition by the RecX regulator protein, and in general displayed a greater persistence on DNA. The enhancement in conjugational function comes at the price of a measurable RecA-mediated cellular growth deficiency. Persistent DNA binding represents a barrier to other processes of DNA metabolism in vivo. The growth deficiency is alleviated by expression of the functionally robust RecX protein from Neisseria gonorrhoeae. RecA filaments can be a barrier to processes like replication and transcription. RecA regulation by RecX protein is important in maintaining an optimal balance between recombination and other aspects of DNA metabolism.
Highlights
A given segment of chromosomal DNA may be subjected to repair, transcription, replication, and recombination, some or all of these processes occurring within a single cell cycle
The genetic recombination systems of bacteria have not evolved for optimal enzymatic function
As recombination and recombination systems can have deleterious effects, these systems have evolved sufficient function to repair a level of DNA double strand breaks typically encountered during replication and cell division
Summary
A given segment of chromosomal DNA may be subjected to repair, transcription, replication, and recombination, some or all of these processes occurring within a single cell cycle. Each of these processes poses real or potential molecular problems for the others, and many sources of genome instability lie at the interfaces [1,2,3,4]. The role of collisions between replication forks and transient template discontinuities created by DNA repair events in the creation of double strand breaks is well appreciated, as is the importance of recombinational DNA repair of those breaks [8,9,10,11,12,13,14,15,16]. The study described here is based on the following premise: (a) recombination systems can have negative impacts on DNA metabolism; (b) for that reason, recombinases such as RecA have not evolved to promote their characteristic DNA pairing and strand exchange activities optimally, but instead reflect an evolutionary compromise between the positive and negative effects of recombination; (c) substantial increases in recombinase functionality should be possible; and (d) since they were not selected during evolution, increases in recombinase functionality may have deleterious effects on cellular DNA metabolism
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