Spontaneous and double-strand break repair-associated quasipalindrome and frameshift mutagenesis in budding yeast: role of mismatch repair.

Although gene conversion (GC) in Saccharomyces cerevisiae is the most error-free way to repair double-strand breaks (DSBs), the mutation rate during homologous recombination is 1000 times greater than during replication. Many mutations involve dissociating a partially- copied strand from its repair template and re-aligning with the same or another template, leading to -1 frameshifts in homonucleotide runs, quasipalindrome (QP)-associated mutations and microhomology-mediated interchromosomal template switches. We studied GC induced by HO endonuclease cleavage at MATα, repaired by an HMR::KI-URA3 donor. We inserted into HMR::KI-URA3 an 18-bp inverted repeat where one arm had a 4-bp insertion. Most GCs yield MAT::KI-ura3::QP + 4 (Ura-) outcomes, but template-switching produces Ura+ colonies, losing the 4-bp insertion. If the QP arm without the insertion is first encountered by repair DNA polymerase and is then (mis)used as a template, the palindrome is perfected. When the QP + 4 arm is encountered first, Ura+ derivatives only occur after second-end capture and second-strand synthesis. QP + 4 mutations are suppressed by mismatch repair (MMR) proteins Msh2, Msh3, and Mlh1, but not Msh6. Deleting Rdh54 significantly reduces QP mutations only when events creating Ura+ occur in the context of a D-loop but not during second-strand synthesis. A similar bias is found with a proofreading-defective DNA polymerase mutation (poI3-01). DSB-induced mutations differed in several genetic requirements from spontaneous events. We also created a + 1 frameshift in the donor, expanding a run of 4 Cs to 5 Cs. Again, Ura+ recombinants markedly increased by disabling MMR, suggesting that MMR acts during GC but favors the unbroken, template strand.

Dissecting quantitative trait nucleotides by saturation genome editing.

Genome editing technologies have the potential to transform our understanding of how genetic variation gives rise to complex traits through the systematic engineering and phenotypic characterization of genetic variants. However, there has yet to be a system with sufficient efficiency, fidelity, and throughput to comprehensively identify causal variants at the genome scale. Here we explored the ability of templated CRISPR editing systems to install natural variants genome-wide in budding yeast. We optimized several approaches to enhance homology-directed repair (HDR) with donor DNA templates, including donor recruitment to target sites, single-stranded donor production by bacterial retrons, and in vivo plasmid assembly. We uncovered unique advantages of each system that we integrated into a single superior system named MAGESTIC 3.0. We used MAGESTIC 3.0 to dissect causal variants residing in 112 quantitative trait loci across 32 environmental conditions, revealing an enrichment for missense variants and loci with multiple causal variants. MAGESTIC 3.0 will facilitate the functional analysis of the genome at single-nucleotide resolution and provides a roadmap for improving template-based genome editing systems in other organisms.

Nonhomologous tails direct heteroduplex rejection and mismatch correction during single-strand annealing in Saccharomyces cerevisiae.

Single-strand annealing (SSA) is initiated when a double strand break (DSB) occurs between two flanking repeated sequences, resulting in a deletion that leaves a single copy of the repeat. We studied budding yeast strains carrying two 200-bp URA3 sequences separated by 2.6 kb of spacer DNA (phage lambda) in which a site-specific DSB can be created by HO or Cas9 endonucleases. Repeat-mediated deletion requires removal of long 3'-ended single-stranded tails (flaps) by Rad1-Rad10 with the assistance of Msh2-Msh3, Saw1 and Slx4. A natural 3% divergence of unequally spaced heterologies between these repeats (designated F and A) causes a significant reduction in the frequency of SSA repair. This decrease is caused by heteroduplex rejection in which mismatches (MMs) in the annealed intermediate are recognized by the MutS (Msh2 and Msh6) components of the MM repair (MMR) pathway coupled to unwinding of the duplex by the Sgs1-Rmi1-Top3 helicase. MutL homologs, Mlh1-Pms1 (MutL), are not required for rejection but play their expected role in mismatch correction. Remarkably, heteroduplex rejection is very low in strains where the divergent repeats were immediately adjacent (Tailless strains) and the DSB was induced by Cas9. These results suggest that the presence of nonhomologous tails strongly stimulates heteroduplex rejection in SSA. DNA sequencing analysis of SSA products from the FA Tailed strain showed a gradient of correction favoring the sequence opposite each 3' end of the annealed strand. Mismatches located in the center of the repair intermediate were corrected by Msh2-Msh6 mediated mismatch correction, while correction of MMs at the extremity of the SSA intermediate often appears to use a different mechanism, possibly by 3' nonhomologous tail removal that includes part of the homologous sequence. In contrast, in FA Tailless strains there was a uniform repair of the MMs across the repeat. A distinctive pattern of correction was found in the absence of MSH2, in both Tailed and Tailless strains, different from the spectrum seen in a msh3Δ msh6Δ double mutant. Previous work has shown that SSA is Rad51-independent but dependent on the strand annealing activity of Rad52. However Rad52 becomes dispensable in a Tailless construct where the DSB is induced by Cas9 or in transformation of a plasmid where SSA occurs in the absence of nonhomologous tails.

Double-strand break repair-associated intragenic deletions and tandem duplications suggest the architecture of the repair replication fork.

Double-strand break (DSB) repair is associated with a 1000-fold increase in mutations compared to normal replication of the same sequences. In budding yeast, repair of an HO endonuclease-induced DSB at the MATα locus can be repaired by using a homologous, heterochromatic HMR::Kl-URA3 donor harboring a transcriptionally silenced URA3 gene, resulting in a MAT::URA3 (Ura+) repair product where URA3 is expressed. Repair-associated ura3- mutations can be selected by resistance to 5-fluoroorotic acid (FOA). Using this system, we find that a major class of mutations are -1 deletions, almost always in homonucleotide runs, but there are few +1 insertions. In contrast, +1 and -1 insertions in homonucleotide runs are nearly equal among spontaneous mutations. Approximately 10% of repair-associated mutations are interchromosomal template switches (ICTS), even though the K. lactis URA3 sequence embedded in HMR is only 72% identical with S. cerevisiae ura3-52 sequences on a different chromosome. ICTS events begin and end in regions of short microhomology, averaging 7 bp. Long microhomologies are favored, but some ICTS junctions are as short as 2 bp. Both repair-associated intragenic deletions (IDs) and tandem duplications (TDs) are recovered, with junctions sharing short stretches of, on average, 6 bp of microhomology. Intragenic deletions are more than 5 times more frequent than TDs. IDs have a mean length of 60 bp, but, surprisingly there are almost no deletions shorter than 25 bp. In contrast, TDs average only 12 bp. The usage of microhomologies among intragenic deletions is not strongly influenced by the degree of adjacent homeology. Together, these data provide a picture of the structure of the repair replication fork. We suggest that IDs and TDs occur within the migrating D-loop in which DNA polymerase δ copies the template, where the 3' end of a partly copied new DNA strand can dissociate and anneal with a single-stranded region of microhomology that lies either in front or behind the 3' end, within the open structure of a migrating D-loop. Our data suggest that ~100 bp ahead of the polymerase is "open," but that part of the repair replication apparatus remains bound in the 25 bp ahead of the newly copied DNA, preventing annealing. In contrast, the template region behind the polymerase appears to be rapidly reannealed, limiting template switching to a very short region.

Prolonged Cell Cycle Arrest in Response to DNA damage in Yeast Requires the Maintenance of DNA Damage Signaling and the Spindle Assembly Checkpoint.

Cells evoke the DNA damage checkpoint (DDC) to inhibit mitosis in the presence of DNA double-strand breaks (DSBs) to allow more time for DNA repair. In budding yeast, a single irreparable DSB is sufficient to activate the DDC and induce cell cycle arrest prior to anaphase for about 12 to 15 hours, after which cells "adapt" to the damage by extinguishing the DDC and resuming the cell cycle. While activation of the DNA damage-dependent cell cycle arrest is well-understood, how it is maintained remains unclear. To address this, we conditionally depleted key DDC proteins after the DDC was fully activated and monitored changes in the maintenance of cell cycle arrest. Degradation of Ddc2ATRIP, Rad9, Rad24, or Rad53CHK2 results in premature resumption of the cell cycle, indicating that these DDC factors are required both to establish and to maintain the arrest. Dun1 is required for establishment, but not maintenance of arrest, whereas Chk1 is required for prolonged maintenance but not for initial establishment of the mitotic arrest. When the cells are challenged with 2 persistent DSBs, they remain permanently arrested. This permanent arrest is initially dependent on the continuous presence of Ddc2 and Rad53; however, after 15 hours both proteins become dispensable. Instead, the continued mitotic arrest is sustained by spindle-assembly checkpoint (SAC) proteins Mad1, Mad2, and Bub2 but not by Bub2's binding partner Bfa1. These data suggest that prolonged cell cycle arrest in response to 2 DSBs is achieved by a handoff from the DDC to specific components of the SAC. Furthermore, the establishment and maintenance of DNA damage-induced cell cycle arrest requires overlapping but different sets of factors.

Structure-forming CAG/CTG repeats interfere with gap repair to cause repeat expansions and chromosome breaks.

Expanded CAG/CTG repeats are sites of DNA damage, leading to repeat length changes. Homologous recombination (HR) is one cause of repeat instability and we hypothesized that gap filling was a driver of repeat instability during HR. To test this, we developed an assay such that resection and ssDNA gap fill-in would occur across a (CAG)70 or (CTG)70 repeat tract. When the ssDNA template was a CTG sequence, there were increased repeat contractions and a fragile site was created leading to large-scale deletions. When the CTG sequence was on the resected strand, resection was inhibited, resulting in repeat expansions. Increased nucleolytic processing by deletion of Rad9, the ortholog of 53BP1, rescued repeat instability and chromosome breakage. Loss of Rad51 increased contractions implicating a protective role for Rad51 on ssDNA. Together, our work implicates structure-forming repeats as an impediment to resection and gap-filling which can lead to mutations and large-scale deletions.

101 years ago: Hermann Muller's remarkable insight.

More than 20 years before DNA was identified as the hereditary material, the Drosophila geneticist, Hermann Muller, envisioned the fundamental principles that such a molecule must have: to be auto-assembling and to be mutable but then again stable. He followed his prescient review of these properties with a remarkable prediction: learning about the hereditary material and its properties would not come from studying Drosophila, but from studying bacteria and their bacteriophages.

Design and Evaluation of [18F]CHDI-650 as a Positron Emission Tomography Ligand to Image Mutant Huntingtin Aggregates.

Therapeutic interventions are being developed for Huntington's disease (HD), a hallmark of which is mutant huntingtin protein (mHTT) aggregates. Following the advancement to human testing of two [11C]-PET ligands for aggregated mHTT, attributes for further optimization were identified. We replaced the pyridazinone ring of CHDI-180 with a pyrimidine ring and minimized off-target binding using brain homogenate derived from Alzheimer's disease patients. The major in vivo metabolic pathway via aldehyde oxidase was blocked with a 2-methyl group on the pyrimidine ring. A strategically placed ring-nitrogen on the benzoxazole core ensured high free fraction in the brain without introducing efflux. Replacing a methoxy pendant with a fluoro-ethoxy group and introducing deuterium atoms suppressed oxidative defluorination and accumulation of [18F]-signal in bones. The resulting PET ligand, CHDI-650, shows a rapid brain uptake and washout profile in non-human primates and is now being advanced to human testing.

Cas9-mediated endogenous plasmid loss in Borrelia burgdorferi.

The spirochete Borrelia burgdorferi, which causes Lyme disease, has the most segmented genome among known bacteria. In addition to a linear chromosome, the B. burgdorferi genome contains over 20 linear and circular endogenous plasmids. While many of these plasmids are dispensable under in vitro culture conditions, they are maintained during the natural life cycle of the pathogen. Plasmid-encoded functions are required for colonization of the tick vector, transmission to the vertebrate host, and evasion of host immune defenses. Different Borrelia strains can vary substantially in the type of plasmids they carry. The gene composition within the same type of plasmid can also differ from strain to strain, impeding the inference of plasmid function from one strain to another. To facilitate the investigation of the role of specific B. burgdorferi plasmids, we developed a Cas9-based approach that targets a plasmid for removal. As a proof-of-principle, we showed that targeting wild-type Cas9 to several loci on the endogenous plasmids lp25 or lp28-1 of the B. burgdorferi type strain B31 results in sgRNA-specific plasmid loss even when homologous sequences (i.e., potential sequence donors for DNA recombination) are present nearby. Cas9 nickase versions, Cas9D10A or Cas9H840A, also cause plasmid loss, though not as robustly. Thus, sgRNA-directed Cas9 DNA cleavage provides a highly efficient way to eliminate B. burgdorferi endogenous plasmids that are non-essential in axenic culture.

Repair of mismatched templates during Rad51-dependent Break-Induced Replication.

Using budding yeast, we have studied Rad51-dependent break-induced replication (BIR), where the invading 3' end of a site-specific double-strand break (DSB) and a donor template share 108 bp of homology that can be easily altered. BIR still occurs about 10% as often when every 6th base is mismatched as with a perfectly matched donor. Here we explore the tolerance of mismatches in more detail, by examining donor templates that each carry 10 mismatches, each with different spatial arrangements. Although 2 of the 6 arrangements we tested were nearly as efficient as the evenly-spaced reference, 4 were significantly less efficient. A donor with all 10 mismatches clustered at the 3' invading end of the DSB was not impaired compared to arrangements where mismatches were clustered at the 5' end. Our data suggest that the efficiency of strand invasion is principally dictated by thermodynamic considerations, i.e., by the total number of base pairs that can be formed; but mismatch position-specific effects are also important. We also addressed an apparent difference between in vitro and in vivo strand exchange assays, where in vitro studies had suggested that at a single contiguous stretch of 8 consecutive bases was needed to be paired for stable strand pairing, while in vivo assays using 108-bp substrates found significant recombination even when every 6th base was mismatched. Now, using substrates of either 90 or 108 nt-the latter being the size of the in vivo templates-we find that in vitro D-loop results are very similar to the in vivo results. However, there are still notable differences between in vivo and in vitro assays that are especially evident with unevenly-distributed mismatches. Mismatches in the donor template are incorporated into the BIR product in a strongly polar fashion up to ~40 nucleotides from the 3' end. Mismatch incorporation depends on the 3'→ 5' proofreading exonuclease activity of DNA polymerase δ, with little contribution from Msh2/Mlh1 mismatch repair proteins, or from Rad1-Rad10 flap nuclease or the Mph1 helicase. Surprisingly, the probability of a mismatch 27 nt from the 3' end being replaced by donor sequence was the same whether the preceding 26 nucleotides were mismatched every 6th base or fully homologous. These data suggest that DNA polymerase δ "chews back" the 3' end of the invading strand without any mismatch-dependent cues from the strand invasion structure. However, there appears to be an alternative way to incorporate a mismatch at the first base at the 3' end of the donor.

Euchromatin factors HULC and Set1C affect heterochromatin organization and mating-type switching in fission yeast Schizosaccharomyces pombe.

Mating-type (P or M) of fission yeast Schizosaccharomyces pombe is determined by the transcriptionally active mat1 cassette and is switched by gene conversion using a donor, either mat2 or mat3, located in an adjacent heterochromatin region (mating-type switching; MTS). In the switching process, heterochromatic donors of genetic information are selected based on the P or M cell type and on the action of two recombination enhancers, SRE2 promoting the use of mat2-P and SRE3 promoting the use of mat3-M, leading to replacement of the content of the expressed mat1 cassette. Recently, we found that the histone H3K4 methyltransferase complex Set1C participates in donor selection, raising the question of how a complex best known for its effects in euchromatin controls recombination in heterochromatin. Here, we report that the histone H2BK119 ubiquitin ligase complex HULC functions with Set1C in MTS, as mutants in the shf1, brl1, brl2 and rad6 genes showed defects similar to Set1C mutants and belonged to the same epistasis group as set1Δ. Moreover, using H3K4R and H2BK119R histone mutants and a Set1-Y897A catalytic mutant, we found that ubiquitylation of histone H2BK119 by HULC and methylation of histone H3K4 by Set1C are functionally coupled in MTS. Cell-type biases in MTS in these mutants suggested that HULC and Set1C inhibit the use of the SRE3 recombination enhancer in M cells, thus favoring SRE2 and mat2-P. Consistent with this, imbalanced switching in the mutants was traced to compromised association of the directionality factor Swi6 with the recombination enhancers in M cells. Based on their known effects at other chromosomal locations, we speculate that HULC and Set1C control nucleosome mobility and strand invasion near the SRE elements. In addition, we uncovered distinct effects of HULC and Set1C on histone H3K9 methylation and gene silencing, consistent with additional functions in the heterochromatic domain.

A shattering experience.

Tang et al. (2022) report that the DNA breaks that provoke chromothripsis-the pulverization and dramatic assembly into a rearranged chromosome-are generated by the base excision repair APE1 endonuclease, triggered by removing deoxyinosines that are created in DNA::RNA hybrids.

Quality Control: Maintaining molecular order and preventing cellular chaos.

We asked experts from different fields-from genome maintenance and proteostasis to organelle degradation via ubiquitin and autophagy-"What does quality control mean to you?" Despite their diverse backgrounds, they converge on and discuss the importance of continuous quality control at all levels, context, communication, timing, decisions on whether to repair or remove, and the significance of dysregulated quality control in disease.

DNA replication: the recombination connection.

Failure to complete DNA replication is one of the major sources of genome instability leading to aneuploidy, chromosome breakage, and chromosome rearrangements that are associated with human cancer. One of the surprising revelations of the past decade is that the completion of replication at so-called common fragile sites (CFS) occurs very late in the cell cycle - at mitosis - through a process termed MiDAS (mitotic DNA synthesis). MiDAS is strongly related to another cancer-promoting phenomenon: the activation of alternative lengthening of telomeres (ALT). Our understanding of the mechanisms of ALT and MiDAS in mammalian cells has drawn heavily from recent advances in the study of break-induced replication (BIR), especially in budding yeast. We provide new insights into the BIR, MiDAS, and ALT pathways and their shared similarities.

Determining the kinetics of break-induced replication (BIR) by the assay for monitoring BIR elongation rate (AMBER).

A detailed understanding of how homologous recombination proceeds at the molecular level in vivo requires the ability to detect in real time the appearance of specific intermediates of DNA repair. The most detailed analysis of double-strand break (DSB) repair in eukaryotes has come from the study of budding yeast, using an inducible site-specific HO endonuclease to initiate recombination synchronously in nearly all cells of the population. Polymerase chain reaction (PCR) and chromatin immunoprecipitation (ChIP) methods have been used to visualize the timing of the DSB, its resection by 5' to 3' exonucleases, the binding of the Rad51 recombinase and the pairing of the Rad51 filament with a homologous donor sequence. PCR has also been used to identify the next key step: the initiation of new DNA synthesis to extend the invading stand and copy the donor template. In break-induced replication (BIR), there appears to be a very long delay between strand invasion and this primer extension step. Here we describe an alternative method, an assay for monitoring BIR elongation rate (AMBER) based on digital droplet PCR that yields a much earlier time of initial DNA synthesis. We suggest that previous methods have failed to recover the initial long, single-stranded primer extension product that is readily detected by AMBER.

[11C]CHDI-626, a PET Tracer Candidate for Imaging Mutant Huntingtin Aggregates with Reduced Binding to AD Pathological Proteins.

The expanded polyglutamine-containing mutant huntingtin (mHTT) protein is implicated in neuronal degeneration of medium spiny neurons in Huntington's disease (HD) for which multiple therapeutic approaches are currently being evaluated to eliminate or reduce mHTT. Development of effective and orthogonal biomarkers will ensure accurate assessment of the safety and efficacy of pharmacologic interventions. We have identified and optimized a class of ligands that bind to oligomerized/aggregated mHTT, which is a hallmark in the HD postmortem brain. These ligands are potentially useful imaging biomarkers for HD therapeutic development in both preclinical and clinical settings. We describe here the optimization of the benzo[4,5]imidazo[1,2-a]pyrimidine series that show selective binding to mHTT aggregates over Aß- and/or tau-aggregates associated with Alzheimer's disease pathology. Compound [11C]-2 was selected as a clinical candidate based on its high free fraction in the brain, specific binding in the HD mouse model, and rapid brain uptake/washout in nonhuman primate positron emission tomography imaging studies.

Learning Yeast Genetics from Miro Radman.

Miroslav Radman's far-sighted ideas have penetrated many aspects of our study of the repair of broken eukaryotic chromosomes. For over 35 years my lab has studied different aspects of the repair of chromosomal breaks in the budding yeast, Saccharomyces cerevisiae. From the start, we have made what we thought were novel observations that turned out to have been predicted by Miro's extraordinary work in the bacterium Escherichia coli and then later in the radiation-resistant Dienococcus radiodurans. In some cases, we have been able to extend some of his ideas a bit further.

Single-strand template repair: key insights to increase the efficiency of gene editing.

DNA double-strand breaks (DSBs) pose a serious hazard for the stability of the genome. CRISPR-Cas9-mediated gene editing intentionally creates a site-specific DSB to modify the genomic sequence, typically from an introduced single-stranded DNA donor. However, unlike typical forms of homologous recombination, single-strand template repair (SSTR) is Rad51-independent. Moreover, this pathway is distinct from other previously characterized Rad51-independent processes. Here, we briefly review the work characterizing this pathway, and how these findings can be used to guide and improve current gene editing strategies.

Mechanisms restraining break-induced replication at two-ended DNA double-strand breaks.

DNA synthesis during homologous recombination is highly mutagenic and prone to template switches. Two-ended DNA double-strand breaks (DSBs) are usually repaired by gene conversion with a short patch of DNA synthesis, thus limiting the mutation load to the vicinity of the DSB. Single-ended DSBs are repaired by break-induced replication (BIR), which involves extensive and mutagenic DNA synthesis spanning up to hundreds of kilobases. It remains unknown how mutagenic BIR is suppressed at two-ended DSBs. Here, we demonstrate that BIR is suppressed at two-ended DSBs by proteins coordinating the usage of two ends of a DSB: (i) ssDNA annealing proteins Rad52 and Rad59 that promote second end capture, (ii) D-loop unwinding helicase Mph1, and (iii) Mre11-Rad50-Xrs2 complex that promotes synchronous resection of two ends of a DSB. Finally, BIR is also suppressed when Sir2 silences a normally heterochromatic repair template. All of these proteins are particularly important for limiting BIR when recombination occurs between short repetitive sequences, emphasizing the significance of these mechanisms for species carrying many repetitive elements such as humans.