Chapter Introduction
13: Recombinational DNA Repair and Homologous Recombination
13.1 Recombination as a DNA Repair Process
13.2 Enzymatic Machines in Bacterial Recombinational DNA Repair
13.3 Homologous Recombination in Eukaryotes
13.4 Nonhomologous End Joining
MOMENT OF DISCOVERY
Lorraine Symington
We recently figured out how double-strand DNA breaks (DSBs) are processed in eukaryotic cells, as a first step in generating meiotic crossovers. This story started some years ago, when Jack Szostak showed that DSBs stimulate homologous recombination of DNA and function as natural initiators for recombination during meiosis. Researchers also found that DNA ends produced by DSBs undergo degradation of the 5′-ending strand to produce 3′ single-stranded DNA tails, which are required to initiate homologous recombination. Around the same time, Jim Haber found the same thing to be true for the very similar recombination-based process of mating-type switching in yeast. But how are the 3′ single-stranded tails made?
We had been using genetic and biochemical approaches over a number of years, with the expectation that a single enzyme, probably a specialized nuclease, was responsible. We anticipated that a single mutant in yeast would completely block 3′-tail formation, but we were unsuccessful in our efforts to find it. Then a new student arrived in my lab, Eleni Mimitou, and she hypothesized that helicases might also play an important role. The first helicase-encoding gene she deleted from yeast, SGS1, produced a profound defect in double-strand break processing! The role of Sgs1 at such an early stage of recombination was quite surprising because prior studies had shown a role for it in a late stage of recombination.
Eleni went on to show that the function of Sgs1 is partially redundant with that of a nuclease called Exo1. When she deleted both SGS1 and EXO1 from yeast, the resulting strain produced only partially processed DNA ends during recombination, a result that was obvious from the very first experiment that Eleni looked at. She also found that another protein, Sae2, was required to complete the initial processing of DSBs. These results and other data led us to propose a two-step mechanism for the production of 3′ single-stranded tails. After 15 years of working on recombination, all of these results came together in about six months! These “moments” of discovery in science make all of the struggles in between worthwhile.
—Lorraine Symington, on discovering how DNA ends are processed to initiate DNA recombination
Genetic recombination is the exchange of genetic information between chromosomes or between different chromosomal segments in a single chromosome. Such exchanges occur by several mechanisms. Homologous genetic recombination, often simply termed homologous recombination, encompasses genetic exchanges at sequences that are identical, or nearly so, in both DNA segments involved in the recombination. Any sequence will do, as long as it is shared by the DNAs undergoing an exchange. Chapter 14 explores other forms of recombination, including exchanges that require a specific sequence (site-specific recombination and some forms of transposition) and some that can occur almost randomly (other forms of transposition). The idea of processes with the potential to scramble genetic information may seem incompatible with the DNA replication and repair processes that so thoroughly maintain genomic integrity. But for the most part, the disconnect is illusory. First and foremost, homologous recombination is a DNA repair process, directed at the site of double-strand breaks. At their core, homologous genetic recombination and the most common and accurate form of double-strand break repair, called recombinational DNA repair, are exactly the same process and are carried out by the same enzymes. We begin our discussion by focusing on recombination as a repair process.
A double-strand break (DSB) is the most dangerous of all DNA lesions, and the most lethal if left unrepaired. Double-strand breaks usually arise during DNA replication, when replication forks encounter a single-strand break in a template strand. In DNA metabolism, this is a true show-stopper. Broken DNA ends make it impossible for DNA replication to continue. Double-strand breaks can also arise during exposure to UV light or γ radiation. In mammals, partial deficiencies in DSB repair systems have been linked with a genetic predisposition to many forms of cancer. Two genes most closely associated with an inherited predisposition to human breast cancer, BRCA1 and BRCA2 (encoding breast and ovarian cancer type 1 or type 2 susceptibility proteins), are intimately involved in this repair process. A wide range of human genetic diseases that are characterized by genomic instability, developmental abnormalities, light sensitivity, as well as cancer predisposition, have been traced to defects in additional genes involved in homologous recombination and recombinational DNA repair. Like the human DNA repair enzymes described in Chapter 12, the proteins involved in recombinational DNA repair are genomic guardians. If a mammalian embryo completely lacks the capacity for repairing DSBs, that embryo is never born. Its cells divide a few times and then die, the genome reduced to fragments arising from countless failed attempts to repair stalled replication forks. The capacity for the enzymatic repair of DSBs is inherent to every free-living organism.
The need to repair replication forks probably fueled the evolution of recombination systems. DNA damage is common. Oxygen first appeared in the atmosphere 2.3 billion years ago as photosynthesis evolved. However, the advantages of aerobic metabolism could not be fully realized until cells also developed the means to deal with oxidative DNA damage. A bacterial cell grown in an oxidative environment suffers more than 1,000 DNA lesions per cell per generation, and a typical mammalian cell, more than 100,000 DNA lesions every 24 hours. This omnipresent spontaneous DNA damage may have limited the size of a genome that could be replicated successfully in an aerobic organism, until the advent of systems for reconstituting and restarting collapsed replication forks.
Homologous recombination and recombinational DNA repair systems now have a broader range of functions in diploid organisms. In eukaryotes, the recombinational DNA repair machinery facilitates the accurate transmission of large chromosomes from one generation to the next, in addition to repairing DSBs. DSBs are introduced in every chromosome during meiosis. The resulting recombination provides a link between replicated sister chromosomes (chromatids) and ensures their accurate segregation at cell division. This same recombinational DNA repair process also produces chromosomal crossovers as a byproduct, exchanging large segments of genetic material between homologous chromosomes—a process that makes a significant contribution to the genetic diversity that fuels evolution. The study of homologous recombination was originally inspired by its effect on inheritance. To a great extent, the genetic recombination and recombinational DNA repair systems in every organism made the development of the entire science of genetics possible.
Cellular recombinational DNA repair systems are co-opted in additional processes, such as those that trigger changes in fungal mating types, allow pathogenic bacteria to evade host immune systems, and sometimes consummate horizontal gene transfer through genetic exchanges between cellular chromosomes and foreign DNA (see Figure 1-11). In other words, although homologous recombination began as a process of DNA repair, it has evolved into a broader mechanism that allows populations of organisms to genetically adapt more quickly to their environment.
The recombinational DNA repair of damaged replication forks is the centerpiece of our discussion. The resurrection of collapsed replication forks represents a fascinating intersection of every aspect of DNA metabolism—replication, repair, and recombination. Our examination of recombination thus begins with replication. We then expand the discussion to include the recombination processes of bacteria and eukaryotes in a variety of contexts, as well as some alternative paths to the repair of double-strand breaks.