Recombinational crossroads: eukaryotic enzymes and the limits of bacterial precedents.

نویسنده

  • M M Cox
چکیده

Biochemical form is shaped by biological function. Biochemical investigation of enzymes involved in eukaryotic homologous genetic recombination now is progressing rapidly, having only recently been inaugurated with the study of Rad51 protein, a homolog of the bacterial RecA protein. A significant new front is opened with the first report on the in vitro activities of a second and meiosis-specific RecA homolog, the human Dmc1 protein (1). Results on Rad51 and Dmc1 to date have yielded a number of surprises in the form of some intriguing and perhaps unanticipated differences with respect to the RecA protein. Pondering biochemical differences must inevitably bring one back to a fundamental question: why do cells recombine DNA? The history of in vitro research into mechanisms of genetic recombination now spans two decades. Until quite recently, bacteria have provided most of the recombination enzymes and most of the biochemical insight. The bacterial RecA protein has been at the center of this activity. Little genetic information is exchanged between bacterial chromosomes without RecA. The RecA protein aligns two DNA molecules, facilitates a strand switch that generates a crossover between them, and promotes migration of the resulting DNA branch. An ever-growing host of additional proteins prepare DNA substrates for RecA action, modulate RecA binding to DNA, or process the branched DNA recombination intermediates created by RecA (2–4). A parallel universe of recombination enzymes appears to exist in eukaryotes. The curtain opened in 1992 with the demonstration that in Rad51 (5) and Dmc1 (6) yeast possessed at least two RecA homologs. Mammals also possess homologs of both proteins, and their investigation has taken some interesting turns. Targeted disruption of mouse rad51 confers an embryonic lethal phenotype and sensitivity to ionizing radiation (7, 8). Several proteins involved in carcinogenesis interact with Rad51, including p53 (9), BRCA1 (10), and BRCA2 (11). These results have lent some urgency to the in vitro investigation of Rad51. Following the pioneering work of Sung (12), this now is being pursued in more than a dozen laboratories worldwide. The Dmc1 protein of yeast colocalizes with Rad51 on the zygotene chromosomes (13) and also must play an important role in recombination. The Rad51 proteins of yeast and human appear to be quite similar. They form helical filaments on DNA with a structure similar to that of the RecA protein (14, 15), possess a DNAdependent ATPase activity (12, 15), promote a DNA strand exchange reaction (12, 16–18), and require a single-strand DNA binding protein (RPA) for optimal strand exchange activity (12, 17, 19). Li et al. (1) demonstrate that the human Dmc1 protein possesses a DNA-dependent ATPase activity and promotes a DNA strand exchange requiring levels of Dmc1 consistent with the formation of a filament as the active form. So far, both Rad51 and Dmc1 sound like RecA. However, the differences between RecA and its eukaryotic counterparts are substantial. The DNA strand exchange reactions promoted by the human Rad51 and Dmc1 are much less robust than those promoted by RecA protein (1, 17, 18). ATP is hydrolyzed by all of the eukaryotic proteins at rates from 1 to 2 orders of magnitude slower than RecA protein. If Rad51 and Dmc1 are the eukaryotic equivalents to RecA, evolution seems to have served up some markedly hobbled proteins for use in eukaryotic recombination. Biological paradigms can both aid and hinder a biochemical investigation. In bacteria, the functional paradigm that has shaped most in vitro investigations involving RecA is centered on conjugational recombination. However, a strong case can be made that recombination evolved in bacteria not as a means to exchange genetic information between cells, but as a DNA repair process (4, 20, 21). Recombinational DNA repair operates in a world of DNA strand breaks and gaps and, in the absence of external factors like ionizing radiation, most of these probably are associated with replication forks. If a fork encounters an unrepaired DNA lesion, the lesion is repaired via a recombinational repair pathway by using the RecF, RecO, RecR, and other functions in addition to RecA protein (Fig. 1). If a fork encounters a DNA strand break (e.g., as might exist at a site undergoing excision or mismatch repair), the resulting double-strand break is repaired via a pathway requiring the RecBCD enzyme, RecA, and other proteins (Fig. 1). There are as yet no good estimates as to how often a replication fork is halted by damage, and the probability no doubt varies with growth conditions. However, it almost certainly occurs more often than generally is appreciated. Every Escherichia coli cell suffers 3,000–5,000 DNA lesions per generation, most of which are oxidative lesions (4, 22). Cells lacking RecA exhibit high mortality (which can approach 50% even in the absence of added DNA damaging agents or radiation) (4, 22), much if not all of which may reflect an inability to repair stalled replication forks. Cells lacking RecBCD accumulate high levels of unrepaired double-strand breaks (23), which are likely to represent the normal load of replication-associated breaks handled by the RecBCD repair pathway. These observations alone suggest that every time bidirectional replication is initiated at oriC in a wild-type cell, the chance that one or both replication forks will encounter a situation requiring recombinational DNA repair could be in the range of 10–45%, especially in cells grown aerobically in rich media. A recA culture contains an abundance of dead cells (4), but the fraction of cells with unrepairable stalled replication forks must have an upper limit of 50% in any strain that is capable of increasing cell number with time. If the genome is subjected to an abnormal insult in the form of radiation or an added DNA damaging agent, most cells lacking one of the recombination enzymes simply don’t survive. Recombinational DNA repair provides a highly adaptable and sometimes redundant set of proteins and multiple pathways to deal with the full range of DNA gaps, breaks, and branched structures

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عنوان ژورنال:
  • Proceedings of the National Academy of Sciences of the United States of America

دوره 94 22  شماره 

صفحات  -

تاریخ انتشار 1997