Francesca Storici

In vivo site-directed mutagenesis using oligonucleotides

Functional characterization of the genes of higher eukaryotes has been aided by their expression in model organisms and by analyzing site-specific changes in homologous genes in model systems such as the yeast Saccharomyces cerevisiae. Modifying sequences in yeast or other organisms such that no heterologous material is retained requires in vitro mutagenesis together with subcloning. PCR-based procedures that do not involve cloning are inefficient or require multistep reactions that increase the risk of additional mutations. An alternative approach, demonstrated in yeast, relies on transformation with an oligonucleotide, but the method is restricted to the generation of mutants with a selectable phenotype. Oligonucleotides, when combined with gap repair, have also been used to modify plasmids in yeast; however, this approach is limited by restriction-site availability. We have developed a mutagenesis approach in yeast based on transformation by unpurified oligonucleotides that allows the rapid creation of site-specific DNA mutations in vivo. A two-step, cloning-free process, referred to as delitto perfetto, generates products having only the desired mutation, such as a single or multiple base change, an insertion, a small or a large deletion, or even random mutations. The system provides for multiple rounds of mutation in a window up to 200 base pairs. The process is RAD52 dependent, is not constrained by the distribution of naturally occurring restriction sites, and requires minimal DNA sequencing. Because yeast is commonly used for random and selective cloning of genomic DNA from higher eukaryotes such as yeast artificial chromosomes, the delitto perfetto strategy also provides an efficient way to create precise changes in mammalian or other DNA sequences.

The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast.

In vivo genome manipulation through site-directed mutagenesis and chromosome rearrangements has been hindered by the difficulty in achieving high frequencies of targeting and the intensive labor required to create altered genomes that do not contain any heterologous sequence. Here we describe our approach, referred to as delitto perfetto, that combines the versatility of synthetic oligonucleotides for targeting with the practicality of a general selection system. It provides for an enormously wide variety of genome modifications via homologous recombination. Exceptional high frequencies of mutations are reached when a site-specific double-strand break (DSB) is induced within the locus targeted by the synthetic oligonucleotides. Presented in this chapter is an in-depth description of a series of applications of the delitto perfetto strategy for mutagenesis and chromosome modification both with and without the induction of a DSB, along with the procedures and materials.

Differential Transactivation by the p53 Transcription Factor Is Highly Dependent on p53 Level and Promoter Target Sequence

ABSTRACT Little is known about the mechanisms that regulate differential transactivation by p53. We developed a system in the yeast Saccharomyces cerevisiae that addresses p53 transactivation capacity from 26 different p53 response elements (REs) under conditions where all other factors, such as chromatin, are kept constant. The system relies on a tightly regulated promoter (rheostatable) that can provide for a broad range of p53 expression. The p53 transactivation capacity toward each 20- to 22-bp-long RE could be ranked by using a simple phenotypic assay. Surprisingly, there was as much as a 1,000-fold difference in transactivation. There was no correlation between the functional rank and statistical predictions of binding energy of the REs. Instead we found that the central sequence element in an RE greatly affects p53 transactivation capacity, possibly because of DNA structural properties. Our results suggest that intrinsic DNA binding affinity and p53 protein levels are important contributors to p53-induced differential transactivation. These results are also relevant to understanding the regulation by other families of transcription factors that recognize several sequence-related response elements and/or have tightly regulated expression. We found that p53 had weak activity towards half the apoptotic REs. In addition, p53 alleles associated with familial breast cancer, previously classified as wild type, showed subtle differences in transactivation capacity towards several REs.

Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast

The repair of chromosomal double-strand breaks (DSBs) can be accomplished through homologous recombination in most organisms. We report here that exogenous oligonucleotides can efficiently target for repair a single DSB induced in a chromosome of yeast. The efficiency of recombinational targeting leading to a desired DNA change can be as high as 20% of cells. The DSB was generated either by a regulatable I-SceI endonuclease just before transformation or appeared spontaneously at the site of a long inverted repeat composed of human Alu sequences. The approach used features of our previously described delitto perfetto system for selecting transformants with integrative recombinant oligonucleotides. The DSB repair mediated by pairs of complementary integrative recombinant oligonucleotides was efficient for targeting to homologous sequences that were close to or distant from the DSB and in the presence of a competing homologous chromosome in diploid cells. We also demonstrate that a DSB can strongly stimulate recombination with single-stranded DNA, without strand bias. These findings expand current models of DSB repair. In addition, we establish a high-throughput system for rapid genome-wide modification with oligonucleotides.

RNA-templated DNA repair

RNA can act as a template for DNA synthesis in the reverse transcription of retroviruses and retrotransposons and in the elongation of telomeres. Despite its abundance in the nucleus, there has been no evidence for a direct role of RNA as a template in the repair of any chromosomal DNA lesions, including DNA double-strand breaks (DSBs), which are repaired in most organisms by homologous recombination or by non-homologous end joining. An indirect role for RNA in DNA repair, following reverse transcription and formation of a complementary DNA, has been observed in the non-homologous joining of DSB ends. In the yeast Saccharomyces cerevisiae, in which homologous recombination is efficient, RNA was shown to mediate recombination, but only indirectly through a cDNA intermediate generated by the reverse transcriptase function of Ty retrotransposons in Ty particles in the cytoplasm. Although pairing between duplex DNA and single-strand (ss)RNA can occur in vitro and in vivo, direct homologous exchange of genetic information between RNA and DNA molecules has not been observed. We show here that RNA can serve as a template for DNA synthesis during repair of a chromosomal DSB in yeast. The repair was accomplished with RNA oligonucleotides complementary to the broken ends. This and the observation that even yeast replicative DNA polymerases such as α and δ can copy short RNA template tracts in vitro demonstrate that RNA can transfer genetic information in vivo through direct homologous interaction with chromosomal DNA.

Transcript-RNA-templated DNA recombination and repair

Homologous recombination is a molecular process that has multiple important roles in DNA metabolism, both for DNA repair and genetic variation in all forms of life. Generally, homologous recombination involves the exchange of genetic information between two identical or nearly identical DNA molecules; however, homologous recombination can also occur between RNA molecules, as shown for RNA viruses. Previous research showed that synthetic RNA oligonucleotides can act as templates for DNA double-strand break (DSB) repair in yeast and human cells, and artificial long RNA templates injected in ciliate cells can guide genomic rearrangements. Here we report that endogenous transcript RNA mediates homologous recombination with chromosomal DNA in yeast Saccharomyces cerevisiae. We developed a system to detect the events of homologous recombination initiated by transcript RNA following the repair of a chromosomal DSB occurring either in a homologous but remote locus, or in the same transcript-generating locus in reverse-transcription-defective yeast strains. We found that RNA–DNA recombination is blocked by ribonucleases H1 and H2. In the presence of H-type ribonucleases, DSB repair proceeds through a complementary DNA intermediate, whereas in their absence, it proceeds directly through RNA. The proximity of the transcript to its chromosomal DNA partner in the same locus facilitates Rad52-driven homologous recombination during DSB repair. We demonstrate that yeast and human Rad52 proteins efficiently catalyse annealing of RNA to a DSB-like DNA end in vitro. Our results reveal a novel mechanism of homologous recombination and DNA repair in which transcript RNA is used as a template for DSB repair. Thus, considering the abundance of RNA transcripts in cells, RNA may have a marked impact on genomic stability and plasticity.

Hypermutability of damaged single-strand DNA formed at double-strand breaks and uncapped telomeres in yeast Saccharomyces cerevisiae.

The major DNA repair pathways operate on damage in double-strand DNA because they use the intact strand as a template after damage removal. Therefore, lesions in transient single-strand stretches of chromosomal DNA are expected to be especially threatening to genome stability. To test this hypothesis, we designed systems in budding yeast that could generate many kilobases of persistent single-strand DNA next to double-strand breaks or uncapped telomeres. The systems allowed controlled restoration to the double-strand state after applying DNA damage. We found that lesions induced by UV-light and methyl methanesulfonate can be tolerated in long single-strand regions and are hypermutagenic. The hypermutability required PCNA monoubiquitination and was largely attributable to translesion synthesis by the error-prone DNA polymerase ζ. In support of multiple lesions in single-strand DNA being a source of hypermutability, analysis of the UV-induced mutants revealed strong strand-specific bias and unexpectedly high frequency of alleles with widely separated multiple mutations scattered over several kilobases. Hypermutability and multiple mutations associated with lesions in transient stretches of long single-strand DNA may be a source of carcinogenesis and provide selective advantage in adaptive evolution.

Conservative Repair of a Chromosomal Double-Strand Break by Single-Strand DNA through Two Steps of Annealing

ABSTRACT The repair of chromosomal double-strand breaks (DSBs) is essential to normal cell growth, and homologous recombination is a universal process for DSB repair. We explored DSB repair mechanisms in the yeast Saccharomyces cerevisiae using single-strand oligonucleotides with homology to both sides of a DSB. Oligonucleotide-directed repair occurred exclusively via Rad52- and Rad59-mediated single-strand annealing (SSA). Even the SSA domain of human Rad52 provided partial complementation for a null rad52 mutation. The repair did not involve Rad51-driven strand invasion, and moreover the suppression of strand invasion increased repair with oligonucleotides. A DSB was shown to activate targeting by oligonucleotides homologous to only one side of the break at large distances (at least 20 kb) from the break in a strand-biased manner, suggesting extensive 5′ to 3′ resection, followed by the restoration of resected DNA to the double-strand state. We conclude that long resected chromosomal DSB ends are repaired by a single-strand DNA oligonucleotide through two rounds of annealing. The repair by single-strand DNA can be conservative and may allow for accurate restoration of chromosomal DNAs with closely spaced DSBs.

Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA

Abundant ribonucleotide incorporation in DNA during replication and repair has profound consequences for genome stability, but the global distribution of ribonucleotide incorporation is unknown. We developed ribose-seq, a method for capturing unique products generated by alkaline cleavage of DNA at embedded ribonucleotides. High-throughput sequencing of these fragments in DNA from the yeast Saccharomyces cerevisiae revealed widespread ribonucleotide distribution, with a strong preference for cytidine and guanosine, and identified hotspots of ribonucleotide incorporation in nuclear and mitochondrial DNA. Ribonucleotides were primarily incorporated on the newly synthesized leading strand of nuclear DNA and were present upstream of (G+C)-rich tracts in the mitochondrial genome. Ribose-seq is a powerful tool for the systematic profiling of ribonucleotide incorporation in genomic DNA.

The flexible loop of human FEN1 endonuclease is required for flap cleavage during DNA replication and repair

The conserved, structure‐specific flap endonuclease FEN1 cleaves 5′ DNA flaps that arise during replication or repair. To address in vivo mechanisms of flap cleavage, we developed a screen for human FEN1 mutants that are toxic when expressed in yeast. Two targets were revealed: the flexible loop domain and the catalytic site. Toxic mutants caused G2 arrest and cell death and were unable to repair methyl methanesulfonate lesions. All the mutant proteins retained flap binding. Unlike the catalytic site mutants, which lacked cleavage of any 5′ flaps, the loop mutants exhibited partial ability to cut 5′ flaps when an adjacent single nucleotide 3′ flap was present. We suggest that the flexible loop is important for efficient cleavage through positioning the 5′ flap and the catalytic site.