Sensitized genetic systems and DNA at-risk motif reporters have been developed through several years of investigating genetic risks and synergistic interactions with DNA metabolic defects. Repeat motifs have been identified in many organisms that are at high risk for genetic change in wild-type or mutation-prone cells. They can be a source genomic instability, and in some cases provide """"""""phase changes"""""""" that extend the host range. We surmised from our various experimental approaches that many of these At Risk Motifs (ARMs) can form non-canonical DNA structures that are poor substrates for replication or post-replication repair. This led us to propose that ARMs can be a major source of genome instability. They can also be exploited as tools to address aspects of DNA metabolism. Using a variety of genetic and molecular approaches with yeast, we have characterized several types of ARMs and employed them to address mechanisms of repeat instability. These include small direct repeats separated by up to 100 bases, a motif common in all organisms, long inverted repeats in configurations similar to many arrangements of Alu and LINE sequences in the human genome, homonucleotide runs as short as 8 to 10 bases that can lead to extremely high levels of frameshift mutation and other types of unstable mini- and microsatellites. We have established that defects in several aspects of DNA metabolism greatly exacerbate the instability associated with ARMs, in essence greatly increasing the risk associated with the at-risk motifs. DNA repair, replication and processing of DNA intermediates require coordinated interactions between many proteins. The combination of subtle changes in one or more DNA metabolic acting at ARMs can lead to synergistic increases in genome instability. We employ a variety of sophisticated genetic and biochemical approaches to identify combinations of genes that are important for maintaining genome stability. Our studies over the last several years have concentrated on the interplay between genes involved in DNA replication, double-strand break repair, mismatch repair and base excision repair in maintaining genome stability, particularly at ARM sites. We investigated the role of the Pol delta -exonuclease in the maturation of Okazaki fragments into a single continuous strand. Current models of Okazaki fragment maturation propose concerted strand-displacement by Pol delta__3'-5' degradation by Pol delta exonuclease and the degradation of the displaced strand by the 5'-endonuclease activities of FEN1 and Dna2. Pol delta is required for lagging strand replication in eukaryotes and the nuclease activity was generally considered to be required for correcting errors in replication. In collaboration with the Burgers lab (Washington U), we demonstrated that Exo-deficient Pol delta holoenzymes display increased strand displacement which impedes the maturation of Okazaki fragments in the discontinuous DNA strand. This supports our earlier proposal that the exonuclease of the DNA replicative polymerase Pol delta plays an important role in the maturation of Okazaki fragments. Specifically, we have studied this process using both wild type Pol delta and an exo-deficient mutant of Pol delta with increased capacity for strand displacement. The increased stand displacement was primarily due to increased initiation events rather than elongation rate. In the presence of FEN1, Pol delta-exo- nick-translation is more efficient than wild type Pol delta. The optimal rate of maturation of a model Okazaki fragment in our system required that all factors were present stoichiometrically with DNA, except for DNA ligase which required a 10-fold excess. Under these conditions, nick translation past the RNA/DNA junction prior to ligation occurred for only ~5 nt with wild type Pol delta, and 8-10 nt with the exo- mutant enzyme. No significant role for Dna2 could be demonstrated in this maturation process, except when 5'-flaps of 30 nt were present, as previously demonstrated by others. We propose that the role of Dna2 in the maturation of Okazaki fragments is to rescue in rare cases when strand displacement by Pol delta has gone unaccompanied by the 5'-endonuclease activity of FEN1 and/or 3'-5' exonuclease activity of Pol delta. Complementary genetic experiments support this model. pol3-exo- mutations exhibit strong synergistic interactions with a partial FEN1 defect (rad27-p). These mutants also required the double-strand break (DSB) repair system for growth, suggesting accumulation of DSBs. Overexpression of Dna2 rescues the lethality caused by a DSB repair defect. The systems we developed were also used to develop screens for environmental factors that could inhibit DNA metabolic components that are responsible for preventing the natural occurrence of genome instability, rather than acting directly on DNA. While such a mechanism of mutagenesis had been long suspected, an appropriate system with a wide range of detection of genome stability had not been previously available. A variety of factors are being examined, with initial emphasis on metal ions. The screening systems address effects of metal ions on DNA metabolic processes and various types of DNA repair. We found that chronic exposure of yeast to environmentally relevant concentrations of cadmium, a known human carcinogen, can result in extreme hypermutability, as much as 2000-fold. The target of cadmium is the mismatch repair system, rather than DNA, so that naturally occurring replication errors remain uncorrected. Observations with mammalian cell extracts, demonstrate that the observations in yeast may be relevant to humans and suggest a novel mechanism for genome instability. We also performed a genome wide screen in yeast for the genes involved in the repair of aberrant replication intermediates (breaks, stalled forks, etc.). We evaluated the candidate genes for their effects on sensitivity to DNA damage, prevention of hyper-instability caused by at-risk motifs (Alu-inverted repeats)(see ref. 24 in Activities) and viability in combination with a DNA repair defecive rad27/fen1 mutant. A defect in RAD27 (FEN1) leads to impediment of Okazaki maturation and breaks in replicating DNA. In addition to the identification of several known genetic factors, our screen revealed that a defect in BDF1 (bromodomain protein that protects acetylated tails of histone H4) leads to strong synthetic lethality with a rad27/fen1 defect. At the same time, a defect in BDF1 can be tolerated by yeast with a defective DNA polymerase delta that also can lead to DSBs in replicating DNA. These results suggest a special role for histone acetylation in repair or prevention of DNA breaks associated with lagging strand. Once factors are identified in yeast, they can be assessed in vitro using biochemical approaches. Since there is a commonality of repair systems across species, the factors are also investigated in human cells.
Showing the most recent 10 out of 27 publications
