Biomedical subjects

Dilip K Nag

Publications and source records attributed to Dilip K Nag.

6 recordsLinked to original sources

PubMed2006

A region in the dystrophin gene major hot spot harbors a cluster of deletion breakpoints and generates double-strand breaks in yeast.

Deletions within the dystrophin gene (DMD) account for >70% of mutations leading to Duchenne and Becker muscular dystrophies (DMD and BMD). Deletion breakpoints were reported to be scattered within regions that also represent meiotic recombination hot spots. Recent studies indicates that deletion junctions arise from nonhomologous end joining (NHEJ), a major pathway for repairing DNA double-strand breaks (DSBs) in mammals. Here we show that a region in intron 47 (i.e., a major deletion hot spot in the DMD gene) generates DSBs during meiosis in yeast and harbors a cluster of previously sequenced deletion breaks. Mapping of breakpoints in 26 BMD/DMD patients indicated that the frequency of breakpoint occurrence around this region is 3-fold higher than expected by chance. These findings suggest that DSBs mediate deletion formation in intron 47 and possibly account for the high frequency of meiotic recombination in the region. Statistical analysis indicated the presence of at least one other breakpoint cluster in intron 47. Taken together, these results suggest that the primary events in deletion formation occur within discrete regions and that the scattered breakpoint distribution reflects both a variable degree of DSB end processing and the availability of a small (compared to the huge regions involved) deletion junction sample.

Base Sequence↗

PubMed2006

Both conserved and non-conserved regions of Spo11 are essential for meiotic recombination initiation in yeast.

DNA double-strand breaks (DSBs) are the initiators of most meiotic recombination events. In Saccharomyces cerevisiae, at least ten genes are necessary for meiotic DSB formation. However, the molecular roles of these proteins are not clearly understood. The meiosis-specific Spo11 protein, which shows sequence similarity with a subunit of an archaeal topoisomerase, is believed to catalyze the meiotic DSB formation. Spo11 is also required for induction of meiotic DSBs at long inverted repeats and at large trinucleotide repeat tracts. Here we report the isolation and characterization of temperature-sensitive spo11-mutant alleles to better understand how Spo11 functions, and how meiotic DSBs are generated at various recombination hotspots. Analysis of mutation sites of isolated spo11-mutant alleles indicated that both N-terminal and C-terminal non-conserved residues of Spo11 are essential for the protein's function, possibly for interaction with other meiotic DSB enzymes. Several of the mutation sites within the conserved region are predicted to lie on the surface of the protein, suggesting that this region is required for activation of the meiotic initiation complex via protein-protein interaction. In addition to the conditional mutants, we isolated partially recombination-defective mutants; analysis of one of these mutants indicated that Ski8, as observed previously, interacts with Spo11 via the latter's C-terminal residues.

Alleles↗

PubMed2005

Inverted repeat-stimulated sister-chromatid exchange events are RAD1-independent but reduced in a msh2 mutant.

Inverted repeats (IRs) and trinucleotide repeats (TNRs) that have the potential to form secondary structures in vivo are known to cause genome rearrangements. Expansions of TNRs in humans are associated with several neurological disorders. Both IRs and TNRs stimulate spontaneous unequal sister-chromatid exchange (SCE) in yeast. Secondary structure-associated SCE events occur via double-strand break repair. Here we show that the rate of spontaneous IR-stimulated unequal SCE events in yeast is significantly reduced in strains with mutations in the mismatch repair genes MSH2 or MSH3, but unaffected by a mutation in the nucleotide excision-repair gene RAD1. Non-IR-associated unequal SCE events are increased in both MMR- and rad1-mutant cells; however, SCE events for both IR- and non-IR-containing substrates occur at a higher level in the exo1 background. Our results suggest that spontaneous SCE occurs by a template switching mechanism. Like IRs, TNRs have been shown to generate double-strand breaks (DSBs) in yeast. TNR expansions in mice are MSH2-dependent. Since IR-mediated SCE events are reduced in msh2 cells, we propose that TNR expansion mutations arise when DSBs are repaired using the sister or the homolog as a template.

DNA Damage↗

PubMed2004

Both CAG repeats and inverted DNA repeats stimulate spontaneous unequal sister-chromatid exchange in Saccharomyces cerevisiae.

Genomic regions containing trinucleotide repeats (TNRs) are highly unstable, as the repeated sequences exhibit a high rate of mutational change, in which they undergo either a contraction or an expansion of repeat numbers. Although expansion of TNRs is associated with several human genetic diseases, the expansion mechanism is poorly understood. Extensive studies in model organisms have indicated that instability of TNRs occurs by several mechanisms, including replication slippage, DNA repair and recombination. In all models, the formation of secondary structures by disease-associated TNRs is a critical step in the mutation process. In this report, we demonstrate that TNRs and inverted repeats (IRs) both of which have the potential to form secondary structures in vivo, increase spontaneous unequal sister-chromatid exchange (SCE) in vegetatively growing yeast cells. Our results also show that TNR-mediated SCE events are independent of RAD50, MRE11 and RAD51, whereas IR-stimulated SCEs are dependent on the RAD52 epistasis-group genes. We propose that many TNR expansion mutations occur by SCE.

DNA↗

PubMed2003

Trinucleotide repeat expansions: timing is everything.

The expansion of trinucleotide repeats is known to cause a growing number of human diseases. However, the mechanism and timing of expansions are poorly understood. Recent studies indicate that expansion mutations occur by multiple pathways during both meiotic and mitotic divisions, and at various stages of cell division. In addition, mismatch repair proteins play a major part in generating expansions.

DNA↗

PubMed2002

Most meiotic CAG repeat tract-length alterations in yeast are SPO11 dependent.

The expansion of trinucleotide repeat sequences associated with hereditary neurological diseases is believed from earlier studies to be due to errors in DNA replication. However, more recent studies have indicated that recombination may play a significant role in triplet repeat expansion. CAG repeat tracts have been shown to induce double-strand breaks (DSBs) during meiosis in yeast, and DSB formation is dependent on the meiotic recombination machinery. The rate of meiotic instability is several fold higher than mitotic instability. To determine whether DSB repair is responsible for the high rate of repeat tract-length alterations, the frequencies of meiotic repeat-tract instability were compared in wild-type and spo11 mutant strains. In the spo11 background, the rate of meiotic repeat-tract instability remained at the mitotic level, suggesting that meiotic alterations of CAG repeat tracts in yeast occur by the recombination mechanism. Several of these meiotic tract-length alterations are due to DSB repair involving use of the sister chromatid as a template.

Base Sequence↗