The Chromosome Stability Group conducts several ongoing research projects within the Laboratory of Molecular Genetics.
Double-Strand Breaks (DSBs), DNA Repair, At-Risk DNA Motifs (ARMs), Replication-Associated Changes, Checkpoint Controls
The 3-Rs of DNA metabolism—replication, recombination, repair—figure prominently in maintaining genome stability as well as providing opportunities for genomic change. Several approaches have been taken that address their contributions to genome stability and instability.
Novel biological function of the 3' » 5' exonuclease of DNA polymerase δ: For over 30 years, the only biological function attributed to the intrinsic 3' » 5' exonuclease activity present in many DNA polymerases was the correction of errors during DNA synthesis. The exonuclease, which was found to prevent excessive strand displacement in vitro, was proposed to back up the Rad27/Fen1 5'-flap endonuclease to create ligatable nicks between adjacent Okazaki fragments during lagging-strand replication. This would prevent DSBs and assure DNA continuity during replication. Through genetic and enzymatic analyses, the group has been characterizing the switching of nascent strands between the polymerization and the exonuclease sites of DNA polymerase δ. (See Jin et al., 2005.)
Oligonucleotide-targeted genomic modification and double-strand break repair: A system has been developed in yeast called delitto perfetto that mediates rapid in vivo changes in genes and chromosomes. It relies on the insertion of a unique cassette into a gene and its subsequent removal with small oligonucleotides that target a region external to the cassette, thereby creating the desired change(s). Inclusion of an inducible DSB within the cassette stimulated oligonucleotide targeting more than 1000-fold. The high transformation frequencies and versatility of the system provide powerful tools for dissecting mechanisms of DSB repair, as well as rapid genome modification. Recently it was demonstrated that a DSB can be repaired by just a single-strand oligonucleotide and that the mechanism involves a single-strand annealing process. (See Storici et al., 2003, 2006.) Using this system, RNA has been shown for the first time to be able to directly repair a DNA double-strand break (Storici et al., 2007). This has many implications for repair processes and their evolution, gene targeting and evolution of repair.
Common human repeat elements as potential determinants of chromosome instability: The genomes of all organisms contain motifs that are at risk (ARMs) for change, and they are often a source of genetic disease. In humans, the frequently repeated Alu element has been suggested to be a source of instability. Using a yeast-based assay along with an analysis of the Alu repeat distribution in humans, closely spaced inverted Alus were shown to be highly unstable and were sources of DSBs leading to severe chromosome mutations. Inverted repeats lead to a hairpin-capped break, and these breaks may be particularly deleterious because they are difficult to repair. The gene MRE11, associated with a rare repair-defect disease, is required to repair such breaks and has been proposed to be a guardian of the genome against novel DNA hairpin structures. Interestingly, the break associated with the inverted repeat is also a target for single-strand oligonucleotide repair. (See Lobachev et al., 2002.)
Recent investigations have demonstrated that yeast cells exposed to ionizing radiation frequently exhibit chromosome aberrations under conditions of high survival and efficient DSB repair, as shown by a combination of karyotype and microarray analysis. Surprisingly, small retrotransposon sequences are the predominant sites for radiation-induced chromosomal rearrangements. Given the efficiency of rearrangements, DSBs induced in these repeats are suggested to be important sources of large chromosomal changes and evolutionary genome-plasticity (in preparation).
Novel mechanism of mutagenesis: As part of studies of DNA at-risk motifs (ARMs), sensitive genetic reporters were developed in yeast to address mechanisms of mutagenesis. Rather than involving direct action on DNA, mutations could be caused by the inhibition of DNA repair or mutation-avoidance systems. It was discovered that environmental concentrations of cadmium, a common human carcinogen, can act as a strong mutagen by directly inactivating DNA mismatch repair rather than by damaging DNA. (See Jin et al., 2003.)
Chromosomal and DNA breaks and repair: The relationship between DSBs and chromosomal changes is not well understood. Using real-time approaches and following the disposition of ends, DSBs were found to rarely transition into chromosome breaks. However, in the absence of the MRE11/RAD50/XRS2 complex, chromosome breaks become frequent. Real-time changes in chromosomes and the appearance of aberrations are being investigated as a function of cell cycle and genes affecting repair and checkpoint responses. (See Lobachev et al., 2004.) Current efforts are directed toward identifying genes and conditions that influence the transition from a DSB to a chromosome break.
A yeast model for Friedreich’s ataxia reveals important role of mitochondria in protecting against nuclear damage: Mitochondria provide cellular energy. Defects in mitochondrial function have been identified with a variety of human diseases including Friedreich’s ataxia (FA), the most common inherited ataxia disease. FA is associated with a deficiency in the mitochondrial iron-binding protein frataxin. Using a yeast-based system, the absence of frataxin was found to cause chromosome instability via the production of reactive oxygen species, demonstrating a direct role of the mitochondria in protecting the integrity of nuclear DNA. (See Karthikeyan et al., 2003.) These findings in yeast have prompted experiments designed to explore the genomic impact of reduced levels of frataxin in human cells using a combination of RNAi approaches and stress responses.
Functions of Master Regulators of Transcription and Consequences of Mutations
Issues of genome instability are being integrated with phenotypic consequences of mutations as well as polymorphisms in master regulatory genes. Genes that are central to transcriptional networks are expected to be important targets for biological changes.
The p53 master regulatory network: structure, mutations, variation and evolution: Nearly all tumors have alterations in the stress responses coordinated by the tumor suppressor p53, a sequence-specific transcription factor. The p53 master regulatory system provides for many responses to DNA damage including, DNA repair, cell cycle arrest and apoptosis. A "rheostatable" p53 expression system was developed in yeast to address the ability of p53 to act as a sequence-specific transcription factor toward its many target genes that are responsible for cellular growth, death and differentiation. Cancer-associated p53 mutants can exhibit altered transcription-regulation functions as well as complete loss of function. When expressed in human cells, the altered-function mutants exhibit considerable variation in biological outcomes including apoptosis and sensitivity to UV and ionizing radiation. Thus, alterations in master regulatory genes can change the network of genes that are activated and the subsequent phenotypes. (See Resnick & Inga, 2003; Menendez et al., 2006; reviewed in Menendez et al., 2007, in press.)
The universe of p53 target sequences: The p53 transactivation network depends on p53 interactions with target sequence response elements (REs). While a consensus 20 base sequence had been defined that includes two 10-base half sites, almost all REs have been found to contain at least one mismatch. Combining yeast and human cell p53 expression systems, it is now possible to define "rules" for p53 interaction with REs, thereby providing opportunities to identify single-nucleotide polymorphisms (SNPs) at p53-targeted promoter sequences. (See Tomso et al., 2005; Menendez et al., 2006; reviewed in Menendez et al., 2007, in press.) These rules and systems are being used to investigate the functional evolution of REs within the p53 master regulatory network (Jegga et al., in preparation).
Surprisingly, a SNP in an apparent p53 half-site in the promoter of the FLT1 gene was found to integrate the VEGF angiogenesis pathway with the p53 stress response network (see Menendez et al., 2006, PNAS). This finding has now been followed by the demonstration that p53 and estrogen receptors can interact through half-sites to enable transcription. In this case, the estrogen receptor, VEGF and p53 pathways are brought together through a single SNP. These results with functional half sites suggest that the universe of p53 responsive genes is both vast and complex and is leading to further investigations of mechanisms and interactions.
Other master regulators: Based on the p53 system in yeast, the group has addressed the interactions between another master regulator, NKX2.5, and its target sequences. NKX2.5 is a homeodomain transcription factor required for heart development. Its mutants can lead to atrial and ventricular septal defects (See Inga et al., 2005).
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