Expansions and Contractions in a Tandem Repeat Induced by Double-Strand Break Repair

104

100

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 (...

Section: Resultsmentioning

confidence: 99%

“…DSBs are initiators of nearly all meiotic recombination in yeast (40), and most meiotic recombination at the HIS4 locus occurs because of DSBs at the HIS4 promoter (site I, Fig. 1A) (41).…”

Section: Resultsmentioning

confidence: 99%

Meiotic instability of CAG repeat tracts occurs by double-strand break repair in yeast

Jankowski

Nasar

Nag

2000

104

100

“…complementary ssDNA formed at the repeats leads to SSA (28), such that one repeat and the sequences between the repeats are deleted (Fig. 2c).…”

Section: Stable Expression Of Brc-rpa Fusion Proteins In Brca2 Mutantmentioning

confidence: 99%

“…HDR appears to be in competition with a second DSB repair pathway, termed SSA (14,28). When a DSB occurs between sequence repeats and is resected to ssDNA, annealing of the MMC treatments were triplicated except that BRC3RPA and BRC1-2RPA stable cell lines were treated six times.…”

Section: Stable Expression Of Brc-rpa Fusion Proteins In Brca2 Mutantmentioning

confidence: 99%

Suppression of the DNA repair defects of BRCA2-deficient cells with heterologous protein fusions

Saeki

Siaud

Christ

et al. 2006

The BRCA2 tumor suppressor plays an important role in the repair of DNA damage by homologous recombination, also termed homology-directed repair (HDR). Human BRCA2 is 3,418 aa and is composed of several domains. The central part of the protein contains multiple copies of a motif that binds the Rad51 recombinase (the BRC repeat), and the C terminus contains domains that have structural similarity to domains in the ssDNA-binding protein replication protein A (RPA). To gain insight into the role of BRCA2 in the repair of DNA damage, we fused a single (BRC3, BRC4) or multiple BRC motifs to the large RPA subunit. Expression of any of these protein fusions in Brca2 mutant cells substantially improved HDR while suppressing mutagenic repair. A fusion containing a Rad52 ssDNA-binding domain also was active in HDR. Mutations that reduced ssDNA or Rad51 binding impaired the ability of the fusion proteins to function in HDR. The high level of spontaneous chromosomal aberrations in Brca2 mutant cells was largely suppressed by the BRC-RPA fusion proteins, supporting the notion that the primary role of BRCA2 in maintaining genomic integrity is in HDR, specifically to deliver Rad51 to ssDNA. The fusion proteins also restored Rad51 focus formation and cellular survival in response to DNA damaging agents. Because as little as 2% of BRCA2 fused to RPA is sufficient to suppress cellular defects found in Brca2-mutant mammalian cells, these results provide insight into the recently discovered diversity of BRCA2 domain structures in different organisms.double-strand break ͉ mammalian cells ͉ Rad51 ͉ homologous recombination ͉ BRC repeat

“…The former requires genes in the RAD52 epistasis group and relies on extensive homology between the damaged DNA and an undamaged partner [sister chromatid or homologous chromosome (8)]. By contrast, NHEJ requires little or no homology between the recombining molecules (9).…”

mentioning

confidence: 99%

Saccharomyces cerevisiae Sin3p facilitates DNA double-strand break repair

Jazayeri

McAinsh

Jackson

2004

148

114

There are two main pathways in eukaryotic cells for the repair of DNA double-strand breaks: homologous recombination and nonhomologous end joining. Because eukaryotic genomes are packaged in chromatin, these pathways are likely to require the modulation of chromatin structure. One way to achieve this is by the acetylation of lysine residues on the N-terminal tails of histones. Here we demonstrate that Sin3p and Rpd3p, components of one of the predominant histone deacetylase complexes of Saccharomyces cerevisiae, are required for efficient nonhomologous end joining. We also show that lysine 16 of histone H4 becomes deacetylated in the proximity of a chromosomal DNA doublestrand break in a Sin3p-dependent manner. Taken together, these results define a role for the Sin3p͞Rpd3p complex in the modulation of DNA repair.I n eukaryotic cells, the genome is compacted into chromatin.The basic unit of chromatin is the nucleosome, which is composed of the conserved core histones H2A, H2B, H3, and H4. The structure of nucleosomal DNA is further compacted by a range of other factors, including proteins such as the linker histone H1 that binds to DNA between the nucleosome repeats (1). Various DNA-templated processes such as transcription, replication, and DNA repair take place in the context of chromatin and so must involve the manipulation of nucleosomes. One way to achieve this is through the reversible acetylation, phosphorylation, ubiquitination, or ADP-ribosylation of the histone tails that extend to the accessible surface of the nucleosome core particle (2). It has been proposed that combinations of these modifications create a ''histone code'' that is recognized by other proteins to bring about specific downstream events (3).The most extensive body of data has been accumulated for the effect of nucleosome modifications in transcription (4). However, there is growing evidence that there is cross-talk between chromatin and DNA damage response and repair pathways. This is particularly well established for nucleotide excision repair (NER), in which several chromatin remodeling and modification factors have been implicated (ref. 5 and references therein). As discussed further below, there is also evidence for the importance of chromatin modification in the repair of DNA doublestrand breaks (DSBs), which are generated by ionizing radiation and a range of chemotherapeutic agents. Given the highly recombinogenic and cytotoxic nature of such lesions (6, 7), the impact of chromatin on DSB repair is likely to be of considerable biological importance.There are two principle mechanisms for the repair of DNA DSBs: homologous recombination (HR) and nonhomologous end joining (NHEJ). The former requires genes in the RAD52 epistasis group and relies on extensive homology between the damaged DNA and an undamaged partner [sister chromatid or homologous chromosome (8)]. By contrast, NHEJ requires little or no homology between the recombining molecules (9). Key components of the NHEJ machinery in mammals include the Ku70͞Ku80 heterodimer an...