David R. F. Leach

Bacterial Genome Instability

SUMMARY Bacterial genomes are remarkably stable from one generation to the next but are plastic on an evolutionary time scale, substantially shaped by horizontal gene transfer, genome rearrangement, and the activities of mobile DNA elements. This implies the existence of a delicate balance between the maintenance of genome stability and the tolerance of genome instability. In this review, we describe the specialized genetic elements and the endogenous processes that contribute to genome instability. We then discuss the consequences of genome instability at the physiological level, where cells have harnessed instability to mediate phase and antigenic variation, and at the evolutionary level, where horizontal gene transfer has played an important role. Indeed, this ability to share DNA sequences has played a major part in the evolution of life on Earth. The evolutionary plasticity of bacterial genomes, coupled with the vast numbers of bacteria on the planet, substantially limits our ability to control disease.

The sbcC and sbcD genes of Escherichia coli encode a nuclease involved in palindrome inviability and genetic recombination

Background: Long DNA palindromes have the potential to adopt hairpin or cruciform secondary structures that inhibit DNA replication. In Escherichia coli, this palindrome‐mediated inviability results from the activity of the sbcC and sbcD genes, and genetic observations have suggested that they may encode a nuclease. Mutations in these genes also restore the defect in genetic recombination associated with recBC sbcB mutants.

Tethering on the brink: the evolutionarily conserved Mre11–Rad50 complex

Mre11-Rad50 (MR) proteins are encoded by bacteriophage, eubacterial, archeabacterial and eukaryotic genomes, and form a complex with a remarkable protein architecture. This complex is capable of tethering the ends of DNA molecules, possesses a variety of DNA nuclease, helicase, ATPase and annealing activities, and performs a wide range of functions within cells. It is required for meiotic recombination, double-strand break repair, processing of mis-folded DNA structures and maintaining telomere length. This article reviews current knowledge of the structure and enzymatic activities of the MR complex and attempts to integrate biochemical information with the roles of the protein in a cell.

Control of crossing over.

The Holliday junction is a central intermediate in homologous recombination. It consists of a four-way structure that can be resolved by cleavage to give either the crossover or noncrossover products observed. We show here that the formation of these products is controlled by the E. coli resolvasome (RuvABC) in such way that double-strand break repair (DSBR) leads to crossing over and single-strand gap repair (SSGR) does not lead to crossing over. We argue that the positioning of the RuvABC complex and its consequent direction of junction-cleavage is not random. In fact, the action of the RuvABC complex avoids crossing over in the most commonly predicted situations where Holliday junctions are encountered in DNA replication and repair. Our observations suggest that the positioning of the resolvasome may provide a general biochemical mechanism by which cells can control crossing over in recombination.

SbcCD Causes a Double-Strand Break at a DNA Palindrome in the Escherichia coli Chromosome

Long DNA palindromes are sites of genome instability (deletions, amplification, and translocations) in both prokaryotic and eukaryotic cells. In Escherichia coli, genetic evidence has suggested that they are sites of DNA cleavage by the SbcCD complex that can be repaired by homologous recombination. Here we obtain in vivo physical evidence of an SbcCD-induced DNA double-strand break (DSB) at a palindromic sequence in the E. coli chromosome and show that both ends of the break stimulate recombination. Cleavage is dependent on DNA replication, but the observation of two ends at the break argues that cleavage does not occur at the replication fork. Genetic analysis shows repair of the break requires the RecBCD recombination pathway and PriA, suggesting a mechanism of bacterial DNA DSB repair involving the establishment of replication forks.

Overexpression, purification, and characterization of the SbcCD protein from Escherichia coli.

The sbcC and sbcD genes mediate palindrome inviability in Escherichia coli. ThesbcCD operon has been cloned into the plasmid pTrc99A under the control of the strong trc promoter and introduced into a strain carrying a chromosomal deletion of sbcCD. The SbcC and SbcD polypeptides were overexpressed to 6% of total cell protein, and both polypeptides copurified in a four-step purification procedure. Purified SbcCD is a processive double-strand exonuclease that has an absolute requirement for Mn2+ and uses ATP as a preferred energy source. Gel filtration chromatography and sedimentation equilibrium analyses were used to show that the SbcC and SbcD polypeptides dissociate at some stage after purification and that this dissociation is reversed by the addition of Mn2+. We demonstrate that SbcD has the potential to form a secondary structural motif found in a number of protein phosphatases and suggest that it is a metalloprotein that contains the catalytic center of the SbcCD exonuclease.

Repair of DNA Covalently Linked to Protein

A potentially lethal form of DNA/RNA modification, a cleavage complex, occurs when a nucleic acid-processing enzyme that acts via a transient covalent intermediate becomes trapped at its site of action. A number of overlapping pathways act to repair these lesions and many of the enzymes involved are those that catalyze recombinational-repair processes. A protein, Tdp1, has been identified that reverses cleavage-complex formation by specifically hydrolyzing a tyrosyl-DNA phosphodiester bond. The study of these pathways is both interesting and pertinent as they modulate the effectiveness of many antitumor/antibacterial drugs that act by stabilizing cleavage-complexes in vivo.

Escherichia coli sbcC mutants permit stable propagation of DNA replicons containing a long palindrome

Recombinant DNA libraries generated in vitro should in theory contain all of the sequences of the genomes from which they are derived. However, the literature is dotted with reports of sequences that cannot be recovered, are under-represented, or are highly unstable. In particular, long palindromic nucleotide sequences of perfect or near-perfect symmetry are either lethal to the vector or suffer deletions or other rearrangements that remove symmetry [Collins, Cold Spring Harbor Symp. Quant. Biol. 45 (1981) 409-416; Collins et al., Gene 19 (1982) 139-146; Hagan and Warren, Gene 24 (1983) 317-326]. We report here that mutation of a single gene, namely sbcC, can overcome this inviability and allow for the stable propagation of a 571-bp nearly perfect palindrome in Escherichia coli. This has implications for the choice of strains used for the recovery and analysis of cloned nucleotide sequences.

Nucleolytic processing of a protein-bound DNA end by the E. coli SbcCD (MR) complex

SbcCD and other Mre11/Rad50 (MR) complexes are implicated in the metabolism of DNA ends. They cleave ends sealed by hairpin structures and have been postulated to play roles in removing protein bound to DNA termini. Here we provide direct evidence that the Escherichia coli MR complex (SbcCD) removes protein from a protein-bound DNA end by inserting a double-strand break (DSB). These observations indicate a more complex biochemical action than has been assumed previously and argue that this class of protein has the potential to play a direct role in deprotecting protein-bound DNA ends in vivo.

Repair by recombination of DNA containing a palindromic sequence

We report here that homologous recombination functions are required for the viability of Escherichia coli cells maintaining a 240 bp chromosomal inverted repeat (palindromic) sequence. Wild‐type cells can successfully replicate this palindrome but recA, recB or recC mutants carrying the palindrome are unviable. The dependence on homologous recombination for cell viability is overcome in sbcC mutants. Directly repeated copies of the DNA containing the palindrome are rapidly resolved to single copies in wild‐type cells but not in sbcC mutants. Our results suggest that double‐strand breaks introduced at the palindromic DNA sequence by the SbcCD nuclease are repaired by homologous recombination. The repair is conservative and the palindrome is retained in the repaired chromosome. We conclude that SbcCD can attack secondary structures but that repair conserves the DNA sequence with the potential to fold.