The structural flexibility of RNA underlies fundamental biological processes, but there are no methods for exploring the multiple conformations adopted by RNAs in vivo. We developed cross-linking of matched RNAs and deep sequencing (COMRADES) for in-depth RNA conformation capture, and a pipeline for the retrieval of RNA structural ensembles. Using COMRADES, we determined the architecture of the Zika virus RNA genome inside cells, and identified multiple site-specific interactions with human noncoding RNAs.

Highlights d Comprehensive RNA-RNA networks of the SARS-CoV-2 genome and subgenomes inside cells d Long-range structures spanning thousands of bases resulting in dynamic topologies d Multiple site-specific interactions between host and virus RNAs d An arch around the ribosomal frameshifting element is under purifying selection

DNA replication across blocking lesions occurs by translesion DNA synthesis (TLS), involving a multitude of mutagenic DNA polymerases that operate to protect the mammalian genome. Using a quantitative TLS assay, we identified three main classes of TLS in human cells: two rapid and error-free, and the third slow and error-prone. A single gene, REV3L, encoding the catalytic subunit of DNA polymerase f (polf), was found to have a pivotal role in TLS, being involved in TLS across all lesions examined, except for a TT cyclobutane dimer. Genetic epistasis siRNA analysis indicated that discrete two-polymerase combinations with polf dictate error-prone or error-free TLS across the same lesion. These results highlight the central role of polf in both error-prone and error-free TLS in mammalian cells, and show that bypass of a single lesion may involve at least three different DNA polymerases, operating in different two-polymerase combinations.

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Human cells tolerate UV-induced cyclobutane pyrimidine dimers (CPD) by translesion DNA synthesis (TLS), carried out by DNA polymerase , the POLH gene product. A deficiency in DNA polymerase due to germ-line mutations in POLH causes the hereditary disease xeroderma pigmentosum variant (XPV), which is characterized by sunlight sensitivity and extreme predisposition to sunlight-induced skin cancer. XPV cells are UV hypermutable due to the activity of mutagenic TLS across CPD, which explains the cancer predisposition of the patients. However, the identity of the backup polymerase that carries out this mutagenic TLS was unclear. Here, we show that DNA polymerase cooperates with DNA polymerases and to carry out error-prone TLS across a TT CPD. Moreover, DNA polymerases and , but not , protect XPV cells against UV cytotoxicity, independently of nucleotide excision repair. This presents an extreme example of benefit-risk balance in the activity of TLS polymerases, which provide protection against UV cytotoxicity at the cost of increased mutagenic load.carcinogenesis ͉ DNA repair ͉ lesion bypass ͉ replication ͉ ultraviolet T LS is a fundamental mechanism for tolerating DNA damage that has escaped repair, carried out by specialized lowfidelity DNA polymerases, which synthesize across a wide variety of DNA lesions (1). At least 5 TLS DNA polymerases are present in mammals, four of which, DNA polymerases , , , and REV1, belong to the Y superfamily. The fifth TLS polymerase is pol, which belongs to the B family, and is the only TLS polymerase known to be essential in mammals (2-5). TLS polymerases exhibit a certain degree of specificity for their substrate DNA lesions, and their activity is tightly regulated (6-9). The most well characterized TLS polymerase is pol, which is specialized to bypass cyclobutane pyrimidine dimers (CPD) in a relatively error-free manner. The biological significance of pol is illustrated by the hereditary disease xeroderma pigmentosum variant (XPV), in which germ-line mutations in the POLH gene, encoding pol, cause an extreme 1000-fold increased predisposition to sunlight-induced skin cancer (10, 11). Cells from XPV patients exhibit a slightly increased UV sensitivity, and a dramatic UV hypermutability (12), which is responsible for their extreme cancer predisposition. The UV hypermutability is explained by the activity of a back-up DNA polymerase that performs TLS across CPD with lower efficiency and higher error-frequency. Although there is evidence that pol is involved in TLS across CPD in XPV cells (13-15), additional polymerases may be involved, and the picture is far from being complete. This is an important issue because these polymerases are likely to be driving sunlight-induced skin carcinogenesis in XPV patients.Here, we show that 3 TLS polymerases, pol, pol, and pol, are involved in TLS across CPD in XPV cells. Moreover, pol and pol, but not pol, also provide protection against UV cytotoxicity, independently of nucleotide excision repair (NER). Results Pol, pol, and pol Are Involved in T...

The Coronaviridae are a family of positive-strand RNA viruses that includes SARS-CoV-2, the etiologic agent of the COVID-19 pandemic. Bearing the largest single-stranded RNA genomes in nature, coronaviruses are critically dependent on long-distance RNA-RNA interactions to regulate the viral transcription and replication pathways. Here we experimentally mapped the in vivo RNA-RNA interactome of the full-length SARS-CoV-2 genome and its subgenomic mRNAs. We uncovered a network of RNA-RNA interactions spanning tens of thousands of nucleotides. These interactions reveal that the viral genome adopts alternative topologies inside cells and undergoes genome cyclization. In addition, the SARS-CoV-2 genome and subgenomic mRNAs engage in different interactions with host RNAs. Most importantly, we discovered a long-range RNA-RNA interaction - the FSE-arch - that encircles the programmed ribosomal frame-shifting element. The FSE-arch is conserved in the related MERS-CoV virus and is under purifying selection. Our findings illuminate RNA-based mechanisms governing replication, discontinuous transcription, and translation of coronaviruses, and will aid future efforts to develop antiviral strategies.

DNA lesions can block replication forks and lead to the formation of single-stranded gaps. These replication complications are mitigated by DNA damage tolerance mechanisms, which prevent deleterious outcomes such as cell death, genomic instability, and carcinogenesis. The two main tolerance strategies are translesion DNA synthesis (TLS), in which low-fidelity DNA polymerases bypass the blocking lesion, and homology-dependent repair (HDR; postreplication repair), which is based on the homologous sister chromatid. Here we describe a unique high-resolution method for the simultaneous analysis of TLS and HDR across defined DNA lesions in mammalian genomes. The method is based on insertion of plasmids carrying defined site-specific DNA lesions into mammalian chromosomes, using phage integrase-mediated integration. Using this method we show that mammalian cells use HDR to tolerate DNA damage in their genome. Moreover, analysis of the tolerance of the UV light-induced 6-4 photoproduct, the tobacco smokeinduced benzo[a]pyrene-guanine adduct, and an artificial trimethylene insert shows that each of these three lesions is tolerated by both TLS and HDR. We also determined the specificity of nucleotide insertion opposite these lesions during TLS in human genomes. This unique method will be useful in elucidating the mechanism of DNA damage tolerance in mammalian chromosomes and their connection to pathological processes such as carcinogenesis.error-prone DNA repair | homologous recombination repair | recombinational repair D NA damage is abundant, caused by both external agents such as sunlight and tobacco smoke and intracellular byproducts of metabolism, amounting to about 50,000 lesions per day per cell (1). Despite the presence of effective DNA repair mechanisms that eliminate lesions and restore the original DNA sequence, DNA replication often encounters unrepaired lesions that have escaped repair. These DNA damages may cause arrest of replication forks and the generation of postreplication gaps (2, 3). To complete replication and prevent the formation of double-strand breaks, which are highly deleterious, cells use DNA damage tolerance (DDT) mechanisms. These include translesion DNA synthesis (TLS) and homology-dependent repair (HDR), which enable bypass of the lesions and completion of replication, without removing the lesions from DNA. HDR uses the sequence from the intact sister chromatid to patch the single-stranded template region carrying the lesion. [We term HDR the pathways of DNA damage tolerance that rely on the homologous sister chromatid, also termed postreplication repair (PRR), damage avoidance, template switch, copy choice recombination, and homologous recombination repair.] This is carried out either by physical transfer of the segment complementary to the damaged template [also termed homologous recombination repair (HRR)] or by copying the complementary strand from the sister chromatid (template switch or postreplication repair). TLS employs specialized low-fidelity DNA polymerases to replicate across ...