Question

Homologous Recombination Lecture

Molecular Biology

The ability for yeast and E. coli to do homologous recombination easily

make it very easy to generate specific mutations. To “knock-out” a gene, you need to transform the cells with a PCR product containing an antibiotic resistance cassette flanked by the genomic DNA sequence where you would like to insert the cassette.

Based on our lecture, diagram the process of recombination of the left flank of the following Kanamycin resistance cassette into the E. coli chromosome. Include the Holliday junction formation and resolution. (Assume little or no branch migration)

Answer 1

Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair:

Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in E. coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double- and single-strand linear DNA substrates, so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene tags can be inserted, and regions of bacterial artificial chromosomes (BACs) or the E. coli genome can be subcloned by gene retrieval using recombineering. Most of these constructs can be made within about a week's time.

Recombineering is an in vivo method of genetic engineering used primarily in Escherichia coli that utilizes short 50 base homologies ^1–5. Because recombineering is based on homologous recombination it allows insertion, deletion or alteration of any sequence precisely and is not dependent on the location of restriction sites (Figure 1). Linear DNAs, either double-stranded (ds), usually in the form of PCR products ^{3, 6–8} or single-stranded (ss) synthetic oligonucleotides ^{2, 9} are introduced by electroporation and provide the homologous substrates (i.e. targeting constructs) to create genetic changes. Recombineering is catalyzed by bacteriophage-encoded homologous recombination functions, such as the coliphage λ Red system ³and the RecET system from the Rac prophage ^{4, 10}.

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Figure 1

Overview of bacteriophage l recombination system used for recombineering

Exo has a 5′ to 3′ dsDNA exonuclease activity, which can generate 3′ overhangs on linear DNA. Beta binds the single stranded DNA (3′ overhangs), promotes ss-annealing and generates recombinant DNA. An additional protein, Gam (not shown here), which prevents RecBCD nuclease from degrading double-strand linear DNA fragments, is also required for dsDNA recombineering.

daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation.

TWO-STRAND DNA DAMAGE, RECOMBINATIONAL REPAIR, SOS RESPONSE, AND DNA REPLICATION

Homologous recombination was described in Escherichia coli in the mid-1940s (351), and for many years it was thought to be the result of a sexual process, analogous to that found in eukaryotes. When the sensitivity to DNA damage of the first recombination-deficient mutants was noticed, it was realized that recombination in this bacterium may serve the needs of DNA repair as well (105, 107, 266, 267). Subsequently, genetic studies delineated two recombinational pathways—the primary, RecBC pathway, serving the needs of “sexual” recombination, and the secondary, RecF pathway, kicking in when the primary pathway is inactive and moonlighting at “postreplication repair” of daughter strand gaps (102, 106, 108). Still later, biochemical characterization of recombinational activities suggested that their primary role is in DNA repair (131, 132). Finally, the realization that disintegrated replication forks are reassembled by recombination justified the “repair” purpose for the RecBC pathway (130,333) and prompted a revision of our ideas about the relationships of DNA replication and recombination.

The goal of this review is to consolidate genetic data on homologous recombination, physical data on DNA damage and repair, and biochemical data on recombinational enzymes under a different idea in an attempt to highlight new areas for the future in vitro and in vivo experiments. The different idea is that the primary role of the homologous recombination system in E. coli is to repair lesions associated with DNA replication of damaged template DNA (130,336). Therefore, this review differs from other recent reviews on homologous recombination in E. coli(108, 320,377) in that its two main emphases are on (i) the evidence for recombinational repair in bacteria and (ii) the interactions of various recombinational repair proteins with each other and with the replication machinery. The recombinational repair machinery is conserved among eubacteria, and so the same two basic pathways are present in such dissimilar species as E. coli andBacillus subtilis. Therefore, although concentrating on theE. coli recombinational repair paradigm, occasionally I use evidence from other eubacteria.

Mechanisms of DNA Damage and Repair

Damage reversal and one-strand repair.Bacterial genomic DNA, like any macromolecule, is subject to constant chemical and physical assault. Repair of the resulting lesions is essential if DNA is to serve as the template for transcription and its own reduplication. In the course of evolution, a complex enzymatic machinery has evolved to maintain this centrally important molecule in usable form (195). Repair of some DNA modifications simply reverses the damage, returning DNA directly to its original state. For instance, photolyase, using near UV-visible light, splits UV-induced pyrimidine dimers Another example is the suicidal `

Repair of other types of lesions requires removal of a segment of the DNA strand around the lesion. The double-strandedness of DNA provides the means for repairing the resulting single-strand gaps: the removed bases can be resynthesized by using the intact complementary strand as a template. One example of such a strategy is the repair of modified bases that do not cause DNA distortion. The so-called base excision repair system acts with precision—an enzyme called DNA glycosylase removes a modified base to produce an abasic site, the phosphodiester bond at the 5′ side of the site is broken, and the repair is completed by a single-base nick translation by DNA polymerase (151) and sealing of the nick by DNA ligase. Another repair system, nucleotide excision repair, deals with DNA-distorting lesions. An excinuclease removes a 12- to 13-nucleotide segment of a single strand centered around the lesion, and the resulting gap is filled in by repair synthesis (reviewed in reference 544). The third repair system, methyl-directed mismatch repair, can liberate up to 1,000 nucleotides from one strand in its efforts to correct a single mismatch arising during DNA replication (reviewed in reference440). A lesion affecting a single DNA strand is referred to in this review as one-strand lesion, and repair of such DNA damage is referred to as one-strand repair.

Two-strand repair.Although the bulk of DNA damage affects one strand of a duplex DNA segment, occasionally both DNA strands are damaged opposite each other, resulting in two-strand damage, a term proposed by Howard-Flanders (266). To repair two-strand damage without the loss of sequence information, a cell needs a higher level of redundancy, an extra homologous sequence whose strands could be used to fix both DNA strands of the damaged sequence. The principle of such two-strand repair is depicted in Fig.1. An affected duplex homologously pairs and exchanges strands with an intact homologous duplex (Fig. 1B). The resulting joint molecule is “resolved” by symmetric single-strand cuts in homologous strands, yielding two new DNA molecules, each containing a single one-strand lesion (Fig. 1C). Now the damaged strands can be mended by one-strand repair with the complementary strands as templates

• A “gene knockout” or “knockout” is a mutation that inactivates a gene function.

These mutations are very useful for classical genetic studies as well as for modern techniques including functional genomic

Thus, the strategy of the two-strand repair or to connect or convert in to a two-strand lesion into a pair of one-strand lesions by strand exchange with an intact homologous DNA sequence. Three common phases of the two-strand repair are evident from this scheme. The central phase, during which a damaged DNA sequence trades strands with an intact homologous sequence to form a joint molecule, is called synapsis. In E. coli and other eubacteria, this phase is catalyzed by RecA protein. Accordingly, the preparatory phase preceding the synapsis is called presynapsis, while the resolution of joint molecules is referred to as postsynapsis . The four-strand junctions holding the joint molecules together are usually referred to as Holliday junctions, after Holliday, who recognized their importance in one of the early models of homologous recombination

Compared to the widely used ermF-ermAM cassette, the kanamycin cassette used in the transformation experiments gave rise to additional antibiotic-resistant T. denticola colonies. The kanamycin cassette is effective for allelic replacement mutagenesis as demonstrated by inactivation of two open reading frames of T. denticola, TDE1430 and TDE0911. In addition, the cassette is also functional in trans-chromosomal complementation. This was determined by functional rescue of a periplasmic flagellum (PF)-deficient mutant that had the flgE gene coding for PF hook protein inactivated. The integration of the full-length flgE gene into the genome of the flgE mutant rescued all of the defects associated with the flgE mutant that included the lack of PF filament and spirochetal motility. Taken together, we demonstrate that the kanamycin resistance gene is a suitable cassette for the genetic manipulation of T. denticola that will facilitate the characterization of virulence factors attributed to this important oral pathogen.