SciCombinator

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Concept: Cytosine

65

The RNA World hypothesis presupposes that abiotic reactions originally produced nucleotides, the monomers of RNA and universal constituents of metabolism. However, compatible prebiotic reactions for the synthesis of complementary (that is, base pairing) nucleotides and mechanisms for their mutual selection within a complex chemical environment have not been reported. Here we show that two plausible prebiotic heterocycles, melamine and barbituric acid, form glycosidic linkages with ribose and ribose-5-phosphate in water to produce nucleosides and nucleotides in good yields. Even without purification, these nucleotides base pair in aqueous solution to create linear supramolecular assemblies containing thousands of ordered nucleotides. Nucleotide anomerization and supramolecular assemblies favour the biologically relevant β-anomer form of these ribonucleotides, revealing abiotic mechanisms by which nucleotide structure and configuration could have been originally favoured. These findings indicate that nucleotide formation and selection may have been robust processes on the prebiotic Earth, if other nucleobases preceded those of extant life.

Concepts: DNA, Gene, Genetics, RNA, Chemistry, Nucleoside, Nucleotide, Cytosine

55

The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

Concepts: DNA, Gene, Genetics, Mutation, Genome, RNA, Base pair, Cytosine

31

A detailed understanding of the mechanisms that underlie antibiotic killing is important for the derivation of new classes of antibiotics and clinically useful adjuvants for current antimicrobial therapies. Our efforts to understand why DinB (DNA polymerase IV) overproduction is cytotoxic to Escherichia coli led to the unexpected insight that oxidation of guanine to 8-oxo-guanine in the nucleotide pool underlies much of the cell death caused by both DinB overproduction and bactericidal antibiotics. We propose a model in which the cytotoxicity of beta-lactams and quinolones predominantly results from lethal double-strand DNA breaks caused by incomplete repair of closely spaced 8-oxo-deoxyguanosine lesions, whereas the cytotoxicity of aminoglycosides might additionally result from mistranslation due to the incorporation of 8-oxo-guanine into newly synthesized RNAs.

Concepts: DNA, Gene, Genetics, Cell, Bacteria, Polymerase, Cytosine, Bactericide

27

DNA and RNA oxidation have been linked to diseases such as cancer, arteriosclerosis, neurodegeneration and diabetes. The prototype base modification studied is the 8-hydroxylation of guanine. DNA integrity is maintained by elaborate repair systems, RNA integrity is less studied but relies mainly on degradation.

Concepts: DNA, Gene, Nucleic acid, PH, Adenine, Nucleobase, Guanine, Cytosine

16

We introduce the SPlit-and-conQueR (SPQR) model, a coarse-grained (CG) representation of RNA designed for structure prediction and refinement. In our approach, the representation of a nucleotide consists of a point particle for the phosphate group and an anisotropic particle for the nucleoside. The interactions are, in principle, knowledge-based potentials inspired by the $\mathcal {E}$SCORE function, a base-centered scoring function. However, a special treatment is given to base-pairing interactions and certain geometrical conformations which are lost in a raw knowledge-based model. This results in a representation able to describe planar canonical and non-canonical base pairs and base-phosphate interactions and to distinguish sugar puckers and glycosidic torsion conformations. The model is applied to the folding of several structures, including duplexes with internal loops of non-canonical base pairs, tetraloops, junctions and a pseudoknot. For the majority of these systems, experimental structures are correctly predicted at the level of individual contacts. We also propose a method for efficiently reintroducing atomistic detail from the CG representation.

Concepts: DNA, Genetics, RNA, Base pair, Phosphate, Group, Nucleotide, Cytosine

14

We recently developed base editing, the programmable conversion of target C:G base pairs to T:A without inducing double-stranded DNA breaks (DSBs) or requiring homology-directed repair using engineered fusions of Cas9 variants and cytidine deaminases. Over the past year, the third-generation base editor (BE3) and related technologies have been successfully used by many researchers in a wide range of organisms. The product distribution of base editing-the frequency with which the target C:G is converted to mixtures of undesired by-products, along with the desired T:A product-varies in a target site-dependent manner. We characterize determinants of base editing outcomes in human cells and establish that the formation of undesired products is dependent on uracil N-glycosylase (UNG) and is more likely to occur at target sites containing only a single C within the base editing activity window. We engineered CDA1-BE3 and AID-BE3, which use cytidine deaminase homologs that increase base editing efficiency for some sequences. On the basis of these observations, we engineered fourth-generation base editors (BE4 and SaBE4) that increase the efficiency of C:G to T:A base editing by approximately 50%, while halving the frequency of undesired by-products compared to BE3. Fusing BE3, BE4, SaBE3, or SaBE4 to Gam, a bacteriophage Mu protein that binds DSBs greatly reduces indel formation during base editing, in most cases to below 1.5%, and further improves product purity. BE4, SaBE4, BE4-Gam, and SaBE4-Gam represent the state of the art in C:G-to-T:A base editing, and we recommend their use in future efforts.

Concepts: DNA, Gene, Genetics, Genome, Base pair, Uracil, Cytosine, Uracil-DNA glycosylase

8

APOBEC3H is a deoxycytidine deaminase that can restrict the replication of HIV-1 in the absence of the viral protein Vif that induces APOBEC3H degradation in cells. APOBEC3H exists in humans as seven haplotypes (I-VII) with different cellular stabilities. Of the three stable APOBEC3H haplotypes (II, V, and VII), haplotypes II and V occur most frequently in the population. Despite APOBEC3H being a bona fide restriction factor, there has been no comparative biochemical characterization of APOBEC3H haplotypes. We characterized the single-stranded (ss)DNA scanning mechanisms that haplotypes II and V use to search their ssDNA substrate for cytosine containing deamination motifs. APOBEC3H haplotype II was able to processively deaminate multiple cytosines in a single enzyme-substrate encounter by using sliding, jumping, and intersegmental transfer movements. In contrast, APOBEC3H haplotype V exhibited diminished sliding and intersegmental transfer abilities, but was able to jump along ssDNA. Due to an Asp or Glu at amino acid 178 differentiating these APOBEC3H haplotypes, the data indicated that this amino acid on helix 6 contributes to processivity. The diminished processivity of APOBEC3H haplotype V did not result in a reduced efficiency to restrict HIV-1 replication in single-cycle infectivity assays, suggesting a redundancy in the contributions of jumping and intersegmental transfer to mutagenic efficiency. Optimal processivity on ssDNA also required dimerization of APOBEC3H through the β2 strands. The findings support a model in which jumping can compensate for deficiencies in intersegmental transfer and suggest that APOBEC3H haplotypes II and V induce HIV-1 mutagenesis efficiently, but by different mechanisms.

Concepts: DNA, Protein, Gene, Mutation, Amino acid, DNA replication, Biochemistry, Cytosine

6

The APOBEC3 family of cytidine deaminases cause lethal hypermutation of retroviruses via deamination of newly reverse-transcribed viral DNA. Their ability to bind RNA is essential for virion infiltration and antiviral activity, yet the mechanisms of viral RNA recognition are unknown. By screening naturally occurring, polymorphic, non-human primate APOBEC3H variants for biological and crystallization properties, we obtained a 2.24-Å crystal structure of pig-tailed macaque APOBEC3H with bound RNA. Here, we report that APOBEC3H forms a dimer around a short RNA duplex and, despite the bound RNA, has potent cytidine deaminase activity. The structure reveals an unusual RNA-binding mode in which two APOBEC3H molecules at opposite ends of a seven-base-pair duplex interact extensively with both RNA strands, but form no protein-protein contacts. CLIP-seq analysis revealed that APOBEC3H preferentially binds to sequences in the viral genome predicted to contain duplexes, a property that may facilitate both virion incorporation and catalytic activity.

Concepts: DNA, Gene, Crystal, Virus, RNA, Ribozyme, Primate, Cytosine

5

APOBEC3A and APOBEC3B, cytidine deaminases of the APOBEC family, are among the main factors causing mutations in human cancers. APOBEC deaminates cytosines in single-stranded DNA (ssDNA). A fraction of the APOBEC-induced mutations occur as clusters (“kataegis”) in single-stranded DNA produced during repair of double-stranded breaks (DSBs). However, the properties of the remaining 87% of nonclustered APOBEC-induced mutations, the source and the genomic distribution of the ssDNA where they occur, are largely unknown. By analyzing genomic and exomic cancer databases, we show that >33% of dispersed APOBEC-induced mutations occur on the lagging strand during DNA replication, thus unraveling the major source of ssDNA targeted by APOBEC in cancer. Although methylated cytosine is generally more mutation-prone than nonmethylated cytosine, we report that methylation reduces the rate of APOBEC-induced mutations by a factor of roughly two. Finally, we show that in cancers with extensive APOBEC-induced mutagenesis, there is almost no increase in mutation rates in late replicating regions (contrary to other cancers). Because late-replicating regions are depleted in exons, this results in a 1.3-fold higher fraction of mutations residing within exons in such cancers. This study provides novel insight into the APOBEC-induced mutagenesis and describes the peculiarity of the mutational processes in cancers with the signature of APOBEC-induced mutations.

Concepts: DNA, Gene, Genetics, Mutation, Base pair, DNA replication, Mutagen, Cytosine

4

Deep sequencing technologies are revealing the complexities of cancer evolution, casting light on mutational processes fueling tumor adaptation, immune escape, and treatment resistance. Understanding mechanisms driving cancer diversity is a critical step toward developing strategies to attenuate tumor evolution and adaptation. One emerging mechanism fueling tumor diversity and subclonal evolution is genomic DNA cytosine deamination catalyzed by APOBEC3B and at least one other APOBEC family member. Deregulation of APOBEC3 enzymes causes a general mutator phenotype that manifests as diverse and heterogeneous tumor subclones. Here, we summarize knowledge of the APOBEC DNA deaminase family in cancer, and their role as driving forces for intratumor heterogeneity and a therapeutic target to limit tumor adaptation.

Concepts: Immune system, DNA, Gene, Genetics, Mutation, Evolution, Molecular biology, Cytosine