Joan F. Sterling

Genes required for ionizing radiation resistance in yeast

The ability of Saccharomyces cerevisiae to tolerate ionizing radiation damage requires many DNA-repair and checkpoint genes, most having human orthologs. A genome-wide screen of diploid mutants homozygous with respect to deletions of 3,670 nonessential genes revealed 107 new loci that influence γ-ray sensitivity. Many affect replication, recombination and checkpoint functions. Nearly 90% were sensitive to other agents, and most new genes could be assigned to the following functional groups: chromatin remodeling, chromosome segregation, nuclear pore formation, transcription, Golgi/vacuolar activities, ubiquitin-mediated protein degradation, cytokinesis, mitochondrial activity and cell wall maintenance. Over 50% share homology with human genes, including 17 implicated in cancer, indicating that a large set of newly identified human genes may have related roles in the toleration of radiation damage.

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.

An APOBEC3A hypermutation signature is distinguishable from the signature of background mutagenesis by APOBEC3B in human cancers

Elucidation of mutagenic processes shaping cancer genomes is a fundamental problem whose solution promises insights into new treatment, diagnostic and prevention strategies. Single-strand DNA–specific APOBEC cytidine deaminase(s) are major source(s) of mutation in several cancer types. Previous indirect evidence implicated APOBEC3B as the more likely major mutator deaminase, whereas the role of APOBEC3A is not established. Using yeast models enabling the controlled generation of long single-strand genomic DNA substrates, we show that the mutation signatures of APOBEC3A and APOBEC3B are statistically distinguishable. We then apply three complementary approaches to identify cancer samples with mutation signatures resembling either APOBEC. Strikingly, APOBEC3A-like samples have over tenfold more APOBEC-signature mutations than APOBEC3B-like samples. We propose that APOBEC3A-mediated mutagenesis is much more frequent because APOBEC3A itself is highly proficient at generating DNA breaks, whose repair can trigger the formation of single-strand hypermutation substrates.

Flexibility of Eukaryotic Okazaki Fragment Maturation through Regulated Strand Displacement Synthesis

Okazaki fragment maturation to produce continuous lagging strands in eukaryotic cells requires precise coordination of strand displacement synthesis by DNA polymerase δ (Pol δ) with 5·-flap cutting by FEN1RAD27 endonuclease. Excessive strand displacement is normally prevented by the 3·-exonuclease activity of Pol δ. This core maturation machinery can be assisted by Dna2 nuclease/helicase that processes long flaps. Our genetic studies show that deletion of the POL32 (third subunit of Pol δ) or PIF1 helicase genes can suppress lethality or growth defects of rad27Δ pol3-D520V mutants (defective for FEN1RAD27 and the 3·-exonuclease of Pol δ) that produce long flaps and of dna2Δ mutants that are defective in cutting long flaps. On the contrary, pol32Δ or pif1Δ caused lethality of rad27Δ exo1Δ double mutants, suggesting that Pol32 and Pif1 are required to generate longer flaps that can be processed by Dna2 in the absence of the short flap processing activities of FEN1RAD27 and Exo1. The genetic analysis reveals a remarkable flexibility of the Okazaki maturation machinery and is in accord with our biochemical analysis. In vitro, the generation of short flaps by Pol δ is not affected by the presence of Pol32; however, longer flaps only accumulate when Pol32 is present. The presence of FEN1RAD27 during strand displacement synthesis curtails displacement in favor of flap cutting, thus suggesting an active hand-off mechanism from Pol δ to FEN1RAD27. Finally, RNA-DNA hybrids are more readily displaced by Pol δ than DNA hybrids, thereby favoring degradation of initiator RNA during Okazaki maturation.

The Multiple Biological Roles of the 3′→5′ Exonuclease of Saccharomyces cerevisiae DNA Polymerase δ Require Switching between the Polymerase and Exonuclease Domains

ABSTRACT Until recently, the only biological function attributed to the 3′→5′ exonuclease activity of DNA polymerases was proofreading of replication errors. Based on genetic and biochemical analysis of the 3′→5′ exonuclease of yeast DNA polymerase δ (Pol δ) we have discerned additional biological roles for this exonuclease in Okazaki fragment maturation and mismatch repair. We asked whether Pol δ exonuclease performs all these biological functions in association with the replicative complex or as an exonuclease separate from the replicating holoenzyme. We have identified yeast Pol δ mutants at Leu523 that are defective in processive DNA synthesis when the rate of misincorporation is high because of a deoxynucleoside triphosphate (dNTP) imbalance. Yet the mutants retain robust 3′→5′ exonuclease activity. Based on biochemical studies, the mutant enzymes appear to be impaired in switching of the nascent 3′ end between the polymerase and the exonuclease sites, resulting in severely impaired biological functions. Mutation rates and spectra and synergistic interactions of the pol3-L523X mutations with msh2, exo1, and rad27/fen1 defects were indistinguishable from those observed with previously studied exonuclease-defective mutants of the Pol δ. We conclude that the three biological functions of the 3′→5′ exonuclease addressed in this study are performed intramolecularly within the replicating holoenzyme.

Base Damage within Single-Strand DNA Underlies In Vivo Hypermutability Induced by a Ubiquitous Environmental Agent

Chromosomal DNA must be in single-strand form for important transactions such as replication, transcription, and recombination to occur. The single-strand DNA (ssDNA) is more prone to damage than double-strand DNA (dsDNA), due to greater exposure of chemically reactive moieties in the nitrogenous bases. Thus, there can be agents that damage regions of ssDNA in vivo while being inert toward dsDNA. To assess the potential hazard posed by such agents, we devised an ssDNA–specific mutagenesis reporter system in budding yeast. The reporter strains bear the cdc13-1 temperature-sensitive mutation, such that shifting to 37°C results in telomere uncapping and ensuing 5′ to 3′ enzymatic resection. This exposes the reporter region, containing three closely-spaced reporter genes, as a long 3′ ssDNA overhang. We validated the ability of the system to detect mutagenic damage within ssDNA by expressing a modified human single-strand specific cytosine deaminase, APOBEC3G. APOBEC3G induced a high density of substitutions at cytosines in the ssDNA overhang strand, resulting in frequent, simultaneous inactivation of two reporter genes. We then examined the mutagenicity of sulfites, a class of reactive sulfur oxides to which humans are exposed frequently via respiration and food intake. Sulfites, at a concentration similar to that found in some foods, induced a high density of mutations, almost always as substitutions at cytosines in the ssDNA overhang strand, resulting in simultaneous inactivation of at least two reporter genes. Furthermore, sulfites formed a long-lived adducted 2′-deoxyuracil intermediate in DNA that was resistant to excision by uracil–DNA N-glycosylase. This intermediate was bypassed by error-prone translesion DNA synthesis, frequently involving Pol ζ, during repair synthesis. Our results suggest that sulfite-induced lesions in DNA can be particularly deleterious, since cells might not possess the means to repair or bypass such lesions accurately.

The Drosophila suppressor of sable gene encodes a polypeptide with regions similar to those of RNA-binding proteins.

The nucleotide sequence of the Drosophila melanogaster suppressor of sable [su(s)] gene has been determined. Comparison of genomic and cDNA sequences indicates that an approximately 7,860-nucleotide primary transcript is processed into an approximately 5-kb message, expressed during all stages of the life cycle, that contains an open reading frame capable of encoding a 1,322-amino-acid protein of approximately 150 kDa. The putative protein contains an RNA recognition motif-like region and a highly charged arginine-, lysine-, serine-, aspartic or glutamic acid-rich region that is similar to a region contained in several RNA-processing proteins. In vitro translation of in vitro-transcribed RNA from a complete cDNA yields a product whose size agrees with the size predicted by the open reading frame. Antisera against su(s) fusion proteins recognize the in vitro-translated protein and detect a protein of identical size in the nuclear fractions from tissue culture cells and embryos. The protein is also present in smaller amounts in cytoplasmic fractions of embryos. That the su(s) protein has regions similar in structure to RNA-processing protein is consistent with its known role in affecting the transcript levels of those alleles that it suppresses.

The Transition of Closely Opposed Lesions to Double-Strand Breaks during Long-Patch Base Excision Repair Is Prevented by the Coordinated Action of DNA Polymerase δ and Rad27/Fen1

ABSTRACT DNA double-strand breaks can result from closely opposed breaks induced directly in complementary strands. Alternatively, double-strand breaks could be generated during repair of clustered damage, where the repair of closely opposed lesions has to be well coordinated. Using single and multiple mutants of Saccharomyces cerevisiae (budding yeast) that impede the interaction of DNA polymerase δ and the 5′-flap endonuclease Rad27/Fen1 with the PCNA sliding clamp, we show that the lack of coordination between these components during long-patch base excision repair of alkylation damage can result in many double-strand breaks within the chromosomes of nondividing haploid cells. This contrasts with the efficient repair of nonclustered methyl methanesulfonate-induced lesions, as measured by quantitative PCR and S1 nuclease cleavage of single-strand break sites. We conclude that closely opposed single-strand lesions are a unique threat to the genome and that repair of closely opposed strand damage requires greater spatial and temporal coordination between the participating proteins than does widely spaced damage in order to prevent the development of double-strand breaks.

Damage-induced localized hypermutability

Genome instability continuously presents perils of cancer, genetic disease and death of a cell or an organism. At the same time, it provides for genome plasticity that is essential for development and evolution. We address here the genome instability confined to a small fraction of DNA adjacent to free DNA ends at uncapped telomeres and double-strand breaks. We found that budding yeast cells can tolerate nearly 20 kilobase regions of subtelomeric single-strand DNA that contain multiple UV-damaged nucleotides. During restoration to the double-strand state, multiple mutations are generated by error-prone translesion synthesis. Genome-wide sequencing demonstrated that multiple regions of damage-induced localized hypermutability can be tolerated, which leads to the simultaneous appearance of multiple mutation clusters in the genomes of UV- irradiated cells. High multiplicity and density of mutations suggest that this novel form of genome instability may play significant roles in generating new alleles for evolutionary selection as well as in the incidence of cancer and genetic disease.

Oxidative stress-induced mutagenesis in single-strand DNA occurs primarily at cytosines and is DNA polymerase zeta-dependent only for adenines and guanines

Localized hyper-mutability caused by accumulation of lesions in persistent single-stranded (ss) DNA has been recently found in several types of cancers. An increase in endogenous levels of reactive oxygen species (ROS) is considered to be one of the hallmarks of cancers. Employing a yeast model system, we addressed the role of oxidative stress as a potential source of hyper-mutability in ssDNA by modulation of the endogenous ROS levels and by exposing cells to oxidative DNA-damaging agents. We report here that under oxidative stress conditions the majority of base substitution mutations in ssDNA are caused by erroneous, DNA polymerase (Pol) zeta-independent bypass of cytosines, resulting in C to T transitions. For all other DNA bases Pol zeta is essential for ROS-induced mutagenesis. The density of ROS-induced mutations in ssDNA is lower, compared to that caused by UV and MMS, which suggests that ssDNA could be actively protected from oxidative damage. These findings have important implications for understanding mechanisms of oxidative mutagenesis, and could be applied to development of anticancer therapies and cancer prevention.