Double strand DNA breaks (DSBs) are dangerous events that can result from various causes including environmental assaults or the collapse of DNA replication. While the efficient and precise repair of DSBs is essential for cell survival, faulty repair can lead to genetic instability, making the choice of DSB repair an important step. Here we report that inverted DNA repeats (IRs) placed near a DSB can channel its repair from an accurate pathway that leads to gene conversion to instead a break-induced replication (BIR) pathway that leads to genetic instabilities. The effect of IRs is explained by their ability to form unusual DNA structures when present in ssDNA that is formed by DSB resection. We demonstrate that IRs can form two types of unusual DNA structures, and the choice between these structures depends on the length of the spacer separating IRs. In particular, IRs separated by a long (1-kb) spacer are predominantly involved in inter-molecular single-strand annealing (SSA) leading to the formation of inverted dimers; IRs separated by a short (12-bp) spacer participate in intra-molecular SSA, leading to the formation of fold-back (FB) structures. Both of these structures interfere with an accurate DSB repair by gene conversion and channel DSB repair into BIR, which promotes genomic destabilization. We also report that different protein complexes participate in the processing of FBs containing short (12-bp) versus long (1-kb) ssDNA loops. Specifically, FBs with short loops are processed by the MRX-Sae2 complex, whereas the Rad1-Rad10 complex is responsible for the processing of long loops. Overall, our studies uncover the mechanisms of genomic destabilization resulting from re-routing DSB repair into unusual pathways by IRs. Given the high abundance of IRs in the human genome, our findings may contribute to the understanding of IR-mediated genomic destabilization associated with human disease. Efficient and accurate repair of double-strand DNA breaks (DSBs), resulting from the exposure of cells to ionizing radiation or various chemicals, is crucial for cell survival. Conversely, faulty DSB repair can generate genomic instability that can lead to birth defects or cancer in humans. Here we demonstrate that inverted DNA repeats (IRs) placed in the vicinity of a DSB, interfere with the accurate repair of DSBs and promote genomic rearrangements and chromosome loss. This results from annealing between inverted repeats, located either in different DNA molecules or in the same molecule. In addition, we describe a new role for the Rad1-Rad10 protein complex in processing fold-back (FB) structures formed by intra-molecular annealing involving IRs separated by long spacers. In contrast, FBs with short spacers are processed by the Mre11-Rad50-Xrs2/-Sae2 complex. Overall, we describe several pathways of DSB promoted interaction between IRs that can lead to genomic instability. Given the large number of IRs in the human genome, our findings are relevant to the mechanisms driving genomic destabilization in humans contributing to the development of cancer and other diseases.

The current SARS-CoV-2 pandemic underscores the importance of understanding the evolution of RNA genomes. While RNA is subject to the formation of similar lesions as DNA, the evolutionary and physiological impacts RNA lesions have on viral genomes are yet to be characterized. Lesions that may drive the evolution of RNA genomes can induce breaks that are repaired by recombination or can cause base substitution mutagenesis, also known as base editing. Over the past decade or so, base editing mutagenesis of DNA genomes has been subject to many studies, revealing that exposure of ssDNA is subject to hypermutation that is involved in the etiology of cancer. However, base editing of RNA genomes has not been studied to the same extent. Recently hypermutation of single-stranded RNA viral genomes have also been documented though its role in evolution and population dynamics. Here, we will summarize the current knowledge of key mechanisms and causes of RNA genome instability covering areas from the RNA world theory to the SARS-CoV-2 pandemic of today. We will also highlight the key questions that remain as it pertains to RNA genome instability, mutations accumulation, and experimental strategies for addressing these questions.

Human APOBEC single-strand (ss) specific DNA and RNA cytidine deaminases change cytosines to uracils (U’s) and function in antiviral innate immunity and RNA editing and can cause hypermutation in chromosomes. The resulting U’s can be directly replicated, resulting in C to T mutations, or U–DNA glycosylase can convert the U’s to abasic (AP) sites which are then fixed as C to T or C to G mutations by translesion DNA polymerases. We noticed that in yeast and in human cancers, contributions of C to T and C to G mutations depend on the origin of ssDNA mutagenized by APOBECs. Since ssDNA in eukaryotic genomes readily binds to replication protein A (RPA) we asked if RPA could affect APOBEC-induced mutation spectrum in yeast. For that purpose, we expressed human APOBECs in the wild-type (WT) yeast and in strains carrying a hypomorph mutation rfa1-t33 in the large RPA subunit. We confirmed that the rfa1-t33 allele can facilitate mutagenesis by APOBECs. We also found that the rfa1-t33 mutation changed the ratio of APOBEC3A-induced T to C and T to G mutations in replicating yeast to resemble a ratio observed in long persistent ssDNA in yeast and in cancers. We present the data suggesting that RPA may shield APOBEC formed U’s in ssDNA from Ung1, thereby facilitating C to T mutagenesis through the accurate copying of U’s by replicative DNA polymerases. Unexpectedly, we also found that for U’s shielded from Ung1 by WT RPA, the mutagenic outcome is reduced in the presence of translesion DNA polymerase zeta.

The current SARS- CoV-2 pandemic underscores the importance of understanding the evolution of RNA genomes. While RNA is subject to the formation of similar lesions as DNA, the evolutionary and physiological impacts RNA lesions have on viral genomes are yet to be characterized. Lesions that may drive the evolution of RNA genomes can induce breaks that are repaired by recombination or can cause base substitution mutagenesis, also known as base editing. Over the past decade or so, base editing mutagenesis of DNA genomes has been subject to many studies, revealing that exposure of ssDNA is subject to hypermutation that is involved in the etiology of cancer. However, base editing of RNA genomes has not been studied to the same extent. Recently hypermutation of single-stranded RNA viral genomes have also been documented though its role in evolution and population dynamics. Here, we will summarize the current knowledge of key mechanisms and causes of RNA genome instability covering areas from the RNA world theory to the SARS- CoV-2 pandemic of today. We will also highlight the key questions that remain as it pertains to RNA genome instability, mutations accumulation, and experimental strategies for addressing these questions.

APOBEC cytidine deaminases can convert cytosines to uracils in DNA as well as in RNA. The knowledge of DNA deamination motifs preferred by individual APOBECs revealed APOBEC3A as a major source of hypermutation in cancer. However, the extent and relative contribution of specific APOBECs into RNA editing remains unclear as their preferred RNA-editing motifs have not been defined. Here, using a parallel DNA and RNA sequencing strategy, coupled with motif-centered statistical analyses, we sought to identify mRNA edits and diagnostic editing motifs in yeast and human cells overexpressing individual APOBEC enzymes. This approach revealed a prevailing global enrichment for the uCg trinucleotide motif with even greater preference to the motif’s cytosines located in 3’ base of a loop within a hairpin-loop secondary structure when APOBEC3A, but not any other tested APOBEC, was overexpressed. Further analysis revealed the APOBEC3A-like diagnostic motif enrichment in editing calls from human cancers and blood cells. The APOBEC3A-like editing motif also prevailed in the RNA genomes of SARS-CoV-2 pandemic isolates, as well as in infectious persistent rubella viruses, and in polioviruses emerging from live-attenuated vaccine strains. Together, our results indicate that APOBEC3A is the predominant global APOBEC RNA editor with a potential to impact cell physiology and viral evolution.