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7.11A: Generalized Recombination and RecA

  • Page ID
    9296
  • In homologous recombination, a type of genetic recombination, nucleotide sequences are exchanged between two similar molecules of DNA.

    LEARNING OBJECTIVES

    Explain the process of homologous recombination in bacteria

    KEY TAKEAWAYS

    Key Points

    • Homologous recombination can vary among different organisms and cell types, but most forms involve the same basic steps.
    • Homologous recombination is a major DNA repair process in bacteria. It is also important for producing genetic diversity in bacterial populations.
    • Homologous recombination has been most studied and is best understood for Escherichia coli.

    Key Terms

    • recombination: The formation of genetic combinations in offspring that are not present in the parents
    • genetic: Relating to genetics or genes.
    • homologous: Showing a degree of correspondence or similarity.

    Homologous recombination is a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. It is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks. Homologous recombination also produces new combinations of DNA sequences during meiosis, the process by which eukaryotes make gamete cells, like sperm and egg cells in animals. These new combinations of DNA represent genetic variation in offspring, which in turn enables populations to adapt during the course of evolution. Homologous recombination is also used in horizontal gene transfer to exchange genetic material between different strains and species of bacteria and viruses.

    Homologous recombination can vary among different organisms and cell types, but most forms involve the same basic steps. After a double-strand break occurs, sections of DNA around the 5′ ends of the break are cut away in a process called resection. In the strand invasion step that follows, an overhanging 3′ end of the broken DNA molecule then “invades” a similar or identical DNA molecule that is not broken. After strand invasion, one or two cross-shaped structures called Holliday junctions connect the two DNA molecules. Depending on how the two junctions are cut by enzymes, the type of homologous recombination that occurs in meiosis results in either chromosomal crossover or non-crossover. Homologous recombination that occurs during DNA repair tends to result in non-crossover products, in effect restoring the damaged DNA molecule as it existed before the double-strand break.

    Homologous recombination is conserved across all three domains of life as well as viruses. Homologous recombination is also used in gene targeting, a technique for introducing genetic changes into target organisms.

    Homologous recombination is a major DNA repair process in bacteria. It is also important for producing genetic diversity in bacterial populations. Homologous recombination has been most studied and is best understood for Escherichia coli. Double-strand DNA breaks in bacteria are repaired by the RecBCD pathway of homologous recombination. Breaks that occur on one of the two DNA strands, known as single-strand gaps, are thought to be repaired by the RecF pathway. Both the RecBCD and RecF pathways include a series of reactions known as branch migration, in which single DNA strands are exchanged between two intercrossed molecules of duplex DNA, and resolution, in which those two intercrossed molecules of DNA are cut apart and restored to their normal double-stranded state.

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    Steps in the pre-synapsis phase of homologous recombination in bacteria: Beginning of the RecBCD pathway. This model is based on reactions of DNA and RecBCD with Mg2+ ions in excess over ATP. Step 1: RecBCD binds to a DNA double strand break. Step 2: RecBCD initiates unwinding of the DNA duplex through ATP-dependent helicase activity. Step 3: RecBCD continues its unwinding and moves down the DNA duplex, cleaving the 3′ strand much more frequently than the 5′ strand. Step 4: RecBCD encounters a Chi sequence and stops digesting the 3′ strand; cleavage of the 5′ strand is significantly increased. Step 5: RecBCD loads RecA onto the 3′ strand. Step 6: RecBCD unbinds from the DNA duplex, leaving a RecA nucleoprotein filament on the 3′ tail.

    The RecBCD pathway is the main recombination pathway used in bacteria to repair double-strand breaks in DNA. These double-strand breaks can be caused by UV light and other radiation, as well as chemical mutagens. Double-strand breaks may also arise by DNA replication through a single-strand nick or gap. Such a situation causes what is known as a collapsed replication fork and is fixed by several pathways of homologous recombination including the RecBCD pathway.

    In this pathway, a three-subunit enzyme complex called RecBCD initiates recombination by binding to a blunt or nearly blunt end of a break in double-strand DNA. After RecBCD binds the DNA end, the RecB and RecD subunits begin unzipping the DNA duplex through helicase activity. The RecB subunit also has a nuclease domain, which cuts the single strand of DNA that emerges from the unzipping process. This unzipping continues until RecBCD encounters a specific nucleotide sequence (5′-GCTGGTGG-3′) known as a Chi site.

    Upon encountering a Chi site, the activity of the RecBCD enzyme changes drastically. DNA unwinding pauses for a few seconds and then resumes at roughly half the initial speed. This is likely because the slower RecB helicase unwinds the DNA after Chi, rather than the faster RecD helicase, which unwinds the DNA before Chi. Recognition of the Chi site also changes the RecBCD enzyme so that it cuts the DNA strand with Chi and begins loading multiple RecA proteins onto the single-stranded DNA with the newly generated 3′ end. The resulting RecA-coated nucleoprotein filament then searches out similar sequences of DNA on a homologous chromosome. The search process induces stretching of the DNA duplex, which enhances homology recognition (a mechanism termed conformational proofreading). Upon finding such a sequence, the single-stranded nucleoprotein filament moves into the homologous recipient DNA duplex in a process called strand invasion. The invading 3′ overhang causes one of the strands of the recipient DNA duplex to be displaced, to form a D-loop. If the D-loop is cut, another swapping of strands forms a cross-shaped structure called a Holliday junction.Resolution of the Holliday junction by some combination of RuvABC or RecG can produce two recombinant DNA molecules with reciprocal genetic types, if the two interacting DNA molecules differ genetically. Alternatively, the invading 3′ end near Chi can prime DNA synthesis and form a replication fork. This type of resolution produces only one type of recombinant (non-reciprocal).