The MBNL and CELF proteins act antagonistically to control the alternative splicing of specific exons during mammalian postnatal development. This process is dysregulated in myotonic dystrophy because MBNL proteins are sequestered by (CUG)n and (CCUG)n RNAs expressed from mutant DMPK and ZNF9 genes, respectively. While these observations predict that MBNL proteins have a higher affinity for these pathogenic RNAs versus their normal splicing targets, we demonstrate that MBNL1 possesses comparably high affinities for (CUG)n and (CAG)n RNAs as well as a splicing target, Tnnt3. Mapping of a MBNL1-binding site upstream of the Tnnt3 fetal exon indicates that a preferred binding site for this protein is a GC-rich RNA hairpin containing a pyrimidine mismatch. To investigate how pathogenic RNAs sequester MBNL1 in DM1 cells, we used a combination of chemical/enzymatic structure probing and electron microscopy to determine that MBNL1 forms a ring-like structure which binds to the dsCUG helix. While the MBNL1 N-terminal region is required for RNA binding, the C-terminal region mediates homotypic interactions which may stabilize intra- and/or inter-ring interactions. Our results provide a mechanistic basis for dsCUG-induced MBNL1 sequestration and highlight a striking similarity in the binding sites for MBNL proteins on splicing precursor and pathogenic RNAs.

Individuals with BRCA2 mutations are predisposed to breast cancers owing to genome instability. To determine the functions of BRCA 2, the human protein was purified. It was found to bind selectively to single-stranded DNA (ssDNA), and to ssDNA in tailed duplexes and replication fork structures. Monomeric and dimeric forms of BRCA 2 were observed by EM. BRCA 2 directed the binding of RA D51 recombinase to ssDNA, reduced the binding of RA D51 to duplex DNA and stimulated RA D51-mediated DNA strand exchange. These observations provide a molecular basis for the role of BRCA 2 in the maintenance of genome stability.

Holliday junction (HJ) resolution is essential for chromosome segregation at meiosis and the repair of stalled/collapsed replication forks in mitotic cells. All organisms possess nucleases that promote HJ resolution by the introduction of symmetrically related nicks in two strands at, or close to, the junction point. GEN1, a member of the Rad2/XPG nuclease family, was isolated recently from human cells and shown to promote HJ resolution in vitro and in vivo. Here, we provide the first biochemical/structural characterization of GEN1, showing that, like the Escherichia coli HJ resolvase RuvC, it binds specifically to HJs and resolves them by a dual incision mechanism in which nicks are introduced in the pair of continuous (noncrossing) strands within the lifetime of the GEN1–HJ complex. In contrast to RuvC, but like other Rad2/XPG family members such as FEN1, GEN1 is a monomeric 5′-flap endonuclease. However, the unique feature of GEN1 that distinguishes it from other Rad2/XPG nucleases is its ability to dimerize on HJs. This functional adaptation provides the two symmetrically aligned active sites required for HJ resolution.

Single-strand extensions of the G strand of telomeres are known to be critical for chromosome-end protection and length regulation. Here, we report that in C. elegans, chromosome termini possess 3' G-strand overhangs as well as 5' C-strand overhangs. C tails are as abundant as G tails and are generated by a well-regulated process. These two classes of overhangs are bound by two single-stranded DNA binding proteins, CeOB1 and CeOB2, which exhibit specificity for G-rich or C-rich telomeric DNA. Strains of worms deleted for CeOB1 have elongated telomeres as well as extended G tails, whereas CeOB2 deficiency leads to telomere-length heterogeneity. Both CeOB1 and CeOB2 contain OB (oligo-saccharide/oligo-nucleotide binding) folds, which exhibit structural similarity to the second and first OB folds of the mammalian telomere binding protein hPOT1, respectively. Our results suggest that C. elegans telomere homeostasis relies on a novel mechanism that involves 5' and 3' single-stranded termini.

The replication of long tracts of telomeric repeats may require specific factors to avoid fork regression (Fouché, N., Ö zgür, S., Roy, D., and Griffith, J. (2006) Nucleic Acids Res., in press). Here we show that TRF2 binds to model replication forks and fourway junctions in vitro in a structure-specific but sequence-independent manner. A synthetic peptide encompassing the TRF2 basic domain also binds to DNA four-way junctions, whereas the TRF2 truncation mutant (TRF2 ⌬B ) and a mutant basic domain peptide do not. In the absence of the basic domain, the ability of TRF2 to localize to model telomere ends and facilitate t-loop formation in vitro is diminished. We propose that TRF2 plays a key role during telomere replication in binding chickenfoot intermediates of telomere replication fork regression. Junctionspecific binding would also allow TRF2 to stabilize a strand invasion structure that is thought to exist at the strand invasion site of the t-loop.Telomeres are nucleoprotein structures that protect the ends of chromosomes and are essential for regulating the replicative lifespan of somatic cells. The DNA component of the mammalian telomere consists of long double-stranded (ds) 2 tracts of the hexameric repeat unit TTAGGG (2) that ends with a G-rich 3Ј single-stranded (ss) overhang (3). Telomeric DNA is thought to be organized into a t-loop "end-capping" structure by the telomere-binding proteins TRF1, TRF2, and POT1 and the proteins that bind to them, TIN2, TPP1, and Rap1 (4, 5). This higher order structure may enable cells to distinguish chromosome ends from random double-strand breaks. Large blocks of telomere repeat sequences can be lost when these end-capping proteins are disrupted, or problems are encountered during DNA replication or repair (for review, see Ref. 6). This typically results in p53-and Rb-mediated senescence or cellular crisis, as evidenced by end-to-end fusions of chromosomes, ATM-dependent activation of p53, and apoptosis (for review, see Ref. 7).Much has been learned about the properties of TRF1 and TRF2 including their binding to DNA and the effects of their ablation or overexpression in the cell. We observed that TRF1 forms filamentous structures on long tracts of telomeric DNA in vitro (8), whereas TRF2 binds preferentially to the telomeric DNA at the junction between the duplex repeats and the ss overhang (9). Both TRF1 and TRF2 contain a similar Myb domain at their COOH terminus that mediates their binding to ds telomeric DNA (10). TRF1 and TRF2 differ in their NH 2 termini, however, which are rich in either acidic residues in TRF1 or basic residues in TRF2. The function of the basic domain of TRF2 is poorly understood. Deletion of this domain (TRF2 ⌬B ) does not impede the DNA binding activity of TRF2 or its localization to telomeres in vivo, but expression of TRF2 ⌬B resulted in stochastic deletions of telomeric DNA, generation of t-loop-sized telomeric circles, cell cycle arrest, and induction of senescence in human cells (11,12). In addition, recent evidence suggested that the...