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Expansion of a CGG-repeat tract in the 5' UTR of FMR1 is responsible for the Fragile X-related disorders (FXDs), FXTAS, FXPOI and FXS. Previous work in a mouse model of these disorders has implicated proteins in the base excision and the mismatch repair (MMR) pathways in the expansion mechanism. However, the precise role of these factors in this process is not well understood. The essential role of MutLγ, a complex that plays a minor role in MMR but that is essential for resolving Holliday junctions during meiosis, raises the possibility that expansions proceed via a Holliday junction-like intermediate that is processed to generate a double-strand break (DSB). We show here in an FXD mouse model that LIG4, a ligase essential for non-homologous end-joining (NHEJ), a form of DSB repair (DSBR), protects against expansions. However, a mutation in MRE11, a nuclease that is important for several other DSBR pathways including homologous recombination (HR), has no effect on the extent of expansion. Our results suggest that the expansion pathway competes with NHEJ for the processing of a DSB intermediate. Thus, expansion likely proceeds via an NHEJ-independent DSBR pathway that may also be HR-independent., (Published by Elsevier B.V.)

Friedreich ataxia results from frataxin insufficiency caused by repeat expansion in intron 1 of the frataxin gene. Since the coding sequence is unchanged, the potential exists to ameliorate symptoms by increasing frataxin promoter activity. We therefore defined the minimal frataxin promoter in humans. Despite the fact that frataxin is an essential gene, its promoter is not well conserved in mammals, in part because it has been the frequent target of retroelement insertions. Most of the activity of the human frataxin promoter can be attributed to these retroelements, illustrating how these elements, considered parasitic by some, have been co-opted to drive critical genes. Individuals with the milder French Acadian form and those with the classic form of the disease have no biologically relevant sequence differences in the promoter or 3' UTR, suggesting that some other region of the gene, perhaps the repeat itself, is responsible for the difference in disease severity.

L1 elements are retrotransposons that have been replicating and evolving in mammalian genomes since before the mammalian radiation. Rattus norvegicus shares the young L1mlvi2 clade only with its sister taxon, Rattus cf moluccarius. Here we compared the L1mlvi2 clade in these recently diverged species and found that it evolved rapidly into closely related but distinct clades: the L1mlvi2-rm clade (or subfamily), characterized here from R. cf moluccarius, and the L1mlvi2-rn clade, originally described in R. norvegicus. In addition to other differences, these clades are distinguished by a cluster of amino acid replacement substitutions in ORF I. Both rat species contain the L1mlvi2-rm clade, but the L1mlvi2-rn clade is restricted to R. norvegicus. Therefore, the L1mlvi2-rm clade arose prior to the divergence of R. norvegicus and R. cf moluccarius, and the L1mlvi2-rn clade amplified after their divergence. The total number of L1mlvi2-rm elements in R. cf moluccarius is about the same as the sum of the L1mlvi2-rm and L1mlvi2-rn elements in R. norvegicus. The possibility that L1 amplification is in some way limited so that the two clades compete for replicative supremacy as well as the implications of the other distinguishing characteristic of the L1mlvi2-rn and L1mlvi2-rm clades are discussed.

Tandem repeats are ubiquitous in nature and constitute a major source of genetic variability in populations. This variability is associated with a number of genetic disorders in humans including triplet expansion diseases such as Fragile X syndrome and Huntington's disease. The mechanism responsible for the variability/instability of these tandem arrays remains contentious. We show here that formation of secondary structures, in particular intrastrand tetraplexes, is an intrinsic property of some of the more unstable arrays. Tetraplexes block DNA polymerase progression and may promote instability of tandem arrays by increasing the likelihood of reiterative strand slippage. In the course of doing this work we have shown that some of these tetraplexes involve unusual base interactions. These interactions not only generate tetraplexes with novel properties but also lead us to conclude that the number of sequences that can form stable tetraplexes might be much larger than previously thought.

We have previously shown that the G-rich sequence G16CG(GGT)2GG in the promoter region of the chicken beta-globin gene poses a formidable barrier to DNA synthesis in vitro (Woodford et al., 1994, J. Biol. Chem. 269, 27029-27035). The K+ requirement, template-strand specificity, template concentration independence, and involvement of Hoogsteen bonding suggested that the underlying basis of this new type of DNA synthesis arrest site might be an intrastrand tetrahelical structure. However, the arrest site lacks the four G-rich repeats that are a hallmark of previously described intramolecular tetraplexes and contains a number of noncanonical bases that would be expected to greatly destabilize such a structure. Here we report evidence for an unusual K+-dependent intrastrand "cinched" tetraplex. This structure has several unique features including the incorporation of bases other than guanine into the stem of the tetraplex, interaction between loop bases and bases in the flanking region, and base pairing between bases 3 and 5 of the tetrahelix-forming region to form a molecular "cinch." This finding extends the range of sequences capable of tetraplex formation as well as our appreciation of the conformational complexity of the chicken beta-globin promoter.

We identified and characterized the relics of an ancient rodent L1 family, referred to as Lx, which was extensively amplified at the time of the murine radiation about 12 million years ago, and which we showed was ancestral to the modern L1 families in rat and mouse. Here we have extended our analysis of the Lx amplification by examining more murine and nonmurine species for Lx sequences using both blot hybridization and the polymerase chain reaction for a total of 36 species. In addition we have determined the relative copy number and sequence divergence, or age, of Lx elements in representative murine genera. Our results show that while Lx sequences are confined to murine genera, the extent of the amplification was different in the different murine lineages, indicating that the amplification of Lx did not precede, but was coincident with, the murine radiation. The implications of our findings for the evolutionary dynamics of L1 families and the utility of ancestral amplification events for systematics are discussed.

All modern mammals contain a distinctive, highly repeated (> or = 50,000 members) family of long interspersed repeated DNA called the L1 (LINE 1) family. While the modern L1 families were derived from a common ancestor that predated the mammalian radiation approximately 80 million years ago, most of the members of these families were generated within the last 5 million years. However, recently we demonstrated that modern murine (Old World rats and mice) genomes share an older long interspersed repeated DNA family that we called Lx. Here we report our analysis of the DNA sequence of Lx family members and the relationship of this family to the modern L1 families in mouse and rat. The extent of DNA sequence divergence between Lx members indicates that the Lx amplification occurred about 12 million years ago, around the time of the murine radiation. Parsimony analysis revealed that Lx elements were ancestral to both the modern rat and mouse L1 families. However, we found that few if any of the evolutionary intermediates between the Lx and the modern L1 families were extensively amplified. Because the modern L1 families have evolved under selective pressure, the evolutionary intermediates must have been capable of replication. Therefore, replication-competent L1 elements can reside in genomes without undergoing extensive amplification. We discuss the bearing of our findings on the evolution of L1 DNA elements and the mammalian genome.

We report here that the 64-base pair (bp) guanine-rich polypurine:polypyrimidine tract derived from the right end of the rat long interspersed DNA element is reactive in a supercoil-dependent manner with a variety of chemical probes of non-B DNA structure. At pH 5.0 in the presence of Mg2+, part of the sequence (position 10-40) forms the following two types of triplexes: a G.G.C triplex, and an unusual C.G.C triplex. The latter structure is much more prevalent than the former and is unusual in that the resultant free purine strand forms a hairpin loop. In the absence of Mg2+ the G.G.C triplex disappears and the amount of C.G.C triplex is diminished, and at pH 7.5 in the presence or absence of Mg2+, little or no triplex is observed. Deletion of the 24-bp region just 3' of the triplex-forming region greatly reduces the amount of triplex formed. In this region, which includes an 18-bp polypurine:polypyrimidine sequence, both strands exhibit a moderate symmetric reactivity with the chemical probes tested, independent of pH and Mg2+. The implications of this structurally complex region for the properties of the rat L1 element are discussed.

Repeated DNA sequences were detected as rapidly reannealing sequences in the chromosomal DNA of 13 out of 14 Streptomyces species using either hypochromicity measurements or hydroxyapatite chromatography. These sequences made up between approximately 4% and 11% of the total DNA of these species; only in Streptomyces rimosus were repeated DNA sequences not detected. The repeated sequences fall into a number of distinct percentage G + C (%G + C) classes, many being of rather low %G + C. Analytical density ultracentrifugation of the DNA of these species indicated satellite bands of low %G + C, and high-resolution thermal denaturation profiles indicated the presence of blocks of DNA of low G + C content too. No such satellite band could be found in Streptomyces coelicolor and no low-%G + C DNA could be detected in its thermal denaturation profile. The possible relationship of this repeated DNA, an unusual occurrence in a procaryote, to genetic instability and genetic control mechanisms in Streptomyces is discussed.