Expansion of trinucleotide repeats is associated with a growing number of human diseases. The mechanism and timing of expansion of the repeat tract are poorly understood. In humans, trinucleotide repeats show extreme meiotic instability, and expansion of the repeat tract has been suggested to occur in the germ-line mitotic divisions or postmeiotically during early divisions of the embryo. Studies in model organisms have indicated that polymerase slippage plays a major role in the repeat tract instability and meiotic instability is severalfold higher than the mitotic instability. We show here that meiotic instability of the CAG͞CTG repeat tract in yeast is associated with double-strand break (DSB) formation within the repeated sequences, and that the DSB formation is dependent on the meiotic recombination machinery. The DSB repair results in both expansions and contractions of the CAG repeat tract. E xpansion of trinucleotide repeats has been shown to be associated with a growing number of human diseases (1). Several of these genetic diseases are associated with alterations of the CAG͞CTG (hereafter CAG) repeat tract length. The expansion mutation is highly dependent on the repeat tract length: the longer the repeat tract, the greater the possibility for expansion (2-4). Such mutational changes are dynamic, since the mutated region can undergo further changes in subsequent generations and during the lifespan of an individual.Two mechanisms have been used to explain the expansion of trinucleotide repeats: unequal genetic recombination and error in DNA replication caused by polymerase slippage (5, 6). In the recombination model, unequal crossing-over or gene conversion between triplet repeats either on sister chromatids or on homologs results in an altered tract length. In the slippage model, the replicating strand becomes dissociated, and then it misaligns during reassociation. This misalignment causes the formation of an unpaired sequence either on the template strand or on the nascent strand. A failure to repair the unpaired sequence will result in a DNA molecule containing a larger or smaller number of repeats after the next round of DNA replication. Several studies using model organisms indicate that replication slippage plays a major role in the repeat tract instability (7-14). The latter model is also supported by the in vitro observations that CAG repeats can form hairpin structures, with the CTG hairpin being more stable than the CAG hairpin (15-18). Recent studies with diploid yeast strains containing heterozygous trinucleotide repeat-insertion mutations suggest that trinucleotide repeats in single-stranded DNA are likely to form hairpin structures in vivo (19). During DNA replication, the hairpin formation on the template strand or on the newly synthesized strand would produce a deletion or an expansion of the repeat tract, respectively. This hypothesis is consistent with the observation that CAG-repeat instability depends on the direction in which the replication fork proceeds through the repeat tract (...

SummaryThe genus Mycobacterium includes the major human pathogens Mycobacterium tuberculosis and Mycobacterium leprae. The development of rational drug treatments for the diseases caused by these and other mycobacteria requires the establishment of basic molecular techniques to determine the genetic basis of pathogenesis and drug resistance. To date, the ability to manipulate and move DNA between mycobacterial strains has relied on the processes of transformation and transduction. Here, we describe a naturally occurring conjugation system present in Mycobacterium smegmatis, which we anticipate will further facilitate the ability to manipulate the mycobacterial genome. Our data rule out transduction and transformation as possible mechanisms of gene transfer in this system and are most consistent with conjugal transfer. We show that recombinants are not the result of cell fusion and that transfer occurs from a distinct donor to a recipient. One of the donor strains is mc 2 155, a highly transformable derivative that is considered the prototype laboratory strain for mycobacterial genetics; the demonstration that it is conjugative should increase its genetic manipulability dramatically. During conjugation, extensive regions of chromosomal DNA are transferred into the recipient and then integrated into the recipient chromosome by multiple recombination events. We propose that DNA transfer is occurring by a mechanism similar to Hfr conjugation in Escherichia coli.