Genomic Uracil

The following sections are included:

• List of Contributors
• Acknowledgments
• Contents
• Foreword

Long-term survival of a species requires cellular mechanisms that efficiently safeguard DNA, the chemical material of the genome. All known living organisms have several mechanisms that repair different types of DNA damage. This is essential to prevent cytotoxic and mutagenic effects of damage to DNA. In fact, each cell in the body is inflicted by tens of thousands of DNA lesions per day from spontaneous chemical decay and normal cellular metabolites alone. Environmental chemicals, ultraviolet and ionizing radiation add to the burden of genome damage. Without cellular mechanisms that maintain DNA, we would probably die within a few days from multiple organ failure due to inactivation of numerous genes in each cell. Fortunately, a number of mechanisms contribute to maintaining DNA, including high fidelity DNA replication, DNA repair, cell cycle regulation, as well as removal of cells with serious genetic defects by apoptosis (programmed cell death) (Fig. 1.1). However, this is just the tip of the iceberg, as outlined below.

Life emerged some 3.8 billion years ago, possibly even earlier. Furthermore, in the scenarios for non-enzymatic synthesis of molecules still present in all life forms, uracil was among those found in substantial yields. Uracil may thus have been one of the original bases in RNA polymers in the hypothetical prebiotic RNA World. It may also have been one of the bases in the first cells, which were thought to contain RNA as the hereditary material. The transition to a DNA World had major advantages; the DNA backbone is much more stable than that of RNA because of the reactivity of 2′-hydroxyl group in ribose. This allowed expansion of DNA-genome sizes. Furthermore, RNA replication is intrinsically error-prone compared to DNA replication. DNA cells appear to have completely outcompeted RNA cells. If RNA cells still exist, they must be confined to hitherto unexplored niches. RNA viruses may be remnants of RNA cells, although their origin remains uncertain. The transition from RNA to DNA cells was made possible by evolution of ribonucleotide reductase (RNR, Chapter 3.1.3). This enzyme converts ribonucleotides to deoxyribonucleotides, the building blocks of DNA. The first DNA cells probably contained uracil in the genome, since RNR could directly produce dATP, dGTP, dCTP and dUTP from the corresponding ribonucleotides, but not dTTP. Furthermore, DNAviruses carrying uracil instead of thymine in their genomes do exist, possibly representing relics of a uracil-DNA world…

Uracil in DNA results from incorporation of dUMP instead of dTMP by DNA polymerases during replication, or by spontaneous or enzymatic deamination of already existing DNA cytosines. Which of these that quantitatively dominates in a given cell depends on many factors including cell type and proliferative status, genetic background and exposure to e.g. genotoxic or infectious agents. This chapter will provide an overview of the different routes to genomic uracil, with a major focus on mammalian cells.

Whereas several laboratories contributed to the discovery of other DNA excision repair processes, the discovery of base excision repair (BER) can be uniquely ascribed to the work of Tomas Lindahl (Fig. 4.1). He quantified spontaneous deamination of cytosine to uracil in genomic DNA and reasoned that there had to be a mecshanism correcting the highly mutagenic U:G mismatches resulting from the deamination. The search for such a mechanism resulted in the surprising discovery of uracil-DNA glycosylase in 1974, the first enzyme in a new family of DNA repair proteins. The new enzyme was initially named ‘uracil N-glycosidase,’ but the name was later changed to uracil-DNA glycosylase to comply with current nomenclature. In addition to his work on DNA-cytosine deamination, Lindahl made important discoveries on other aspects of DNA instability, including rates of depurination, depyrimidination and chain breaks at abasic sites…

DNA viruses and retroviruses use various strategies to avoid and remove genomic uracil. Similar to the case for cells, dUTPase and UNG-type uracil-DNA glycosylase are central in these processes. Large DNA viruses encode viral variants of Family 1 (UNG) enzymes, while smaller viruses depend solely on host cell enzymes. Genes for viral dUTPase and UNG are among those that are expressed early after infection. Herpesviridae and poxviridae encode their own uracil-DNA glycosylase, dUTPase and DNA polymerase. Midsize DNA viruses in the adenoviridae family encode their own DNA polymerase, but not uracil-DNA glycosylase. Fowl adenovirus 9 also encodes a dUTPase, but in human adenoviruses a dUTPase-like gene has diverged to become a transforming factor named E4-ORF1. Retroviridae do not encode uracil-DNA glycosylase, but some of them capture host uracil-DNA glycosylase, which is encapsulated in the virus particle and released after infection. Furthermore, several major retroviruses encode dUTPase, although this is not the case for primate lentiviruses that instead use captured UNG to minimize genomic uracil in replicative DNA intermediates. Herpesvirus uracil-DNA glycosylase has strong homology to E. coli, yeast and mammalian UNG proteins, while Poxvirus uracil-DNA glycosylase is a more distant member of the same family. Herpesviruses and adenoviruses replicate in the host nucleus, where they in part can rely on host factors. In contrast, poxviruses replicate in replication factories in the cytosol and encode several DNA replication and repair proteins as well as nucleotide metabolizing enzymes. Interestingly, while herpesvirus and poxvirus UNG proteins are genuine uracil-DNA glycosylases, they also have important functions in viral replication. This has been extensively studied for poxvirus UNG, which is essential for virus replication. Poxvirus UNG (D4) forms a heterotrimeric complex with an adapter protein (A20) and the pox DNA polymerase (E9) and acts as a polymerase processivity factor. Herpes UNG is particularly important for virulence in nerve cells, which lack or have very low levels of cellular UNG. The function of host UNG in retroviridae is complex, as outlined below. The special functions of viral UNG and dUTPase make these proteins potential drug targets. The giant mimivirus Acanthamoeba polyphaga (see Chapter 2) encodes numerous DNA repair proteins, including an UNG-type uracil-DNA glycosylase. Its sequence and crystal structure demonstrate the presence of a catalytic domain containing structural and functional motifs typically present in other UNG proteins and an unstructured N-terminal extension typical of mammalian UNG proteins.

Identification of uracil in DNA as a key intermediate in both adaptive and innate immunity has made research on the introduction and fate of this non-canonical DNA base essential for understanding how we defend ourselves against invading pathogenic microorganisms. All cells have repair systems that remove DNA lesions to preserve genetic integrity. Occasionally, however, some lesions escape repair and result in changes in the DNA sequence. Together with selection of the fittest, this is the concept of evolution of all species. To combat pathogenic bacteria and virus (antigens), specific immune cells (B lymphocytes/B cells) have developed a sophisticated high-speed gene-targeted evolutionary process, termed somatic hypermutation (SHM). This extraordinary mechanism makes B cells capable of producing “tailor-made” high affinity antibodies (immunoglobulins) against any antigen. To initiate SHM, activated B-cells express an enzyme; activation-induced deaminase (AID) that introduces uracil in specific regions of the immunoglobulin (Ig) genes. These uracils are generally not repaired by error free BER, but are processed to generate point mutations or double strand breaks that modulate the antibody binding site or initiate Ig isotype switching, respectively. Cells expressing membrane-bound Igs (B-cell receptors) with increased affinity for antigens are selected to proliferate and differentiate into antibody-secreting plasma cells and memory cells, which combat current infections and protect us against similar future pathogens…

New technologies have made possible sequencing of a large number of whole cancer genomes and exomes. Such studies have identified sequence-associated mutational signatures in human cancers. These frequently suggest a causative mutational agent, thus shedding light on the molecular mechanisms of oncogenesis and cancer progression. Analysis of mutational signatures may also be used to monitor response to therapy…

Correct identification and quantification of uracil levels and location in DNA is essential to understand its biology. Uracil is generally present at very low levels in cellular DNA, so very sensitive analytical methods are necessary for its detection and quantification. Furthermore, small concentrations of contaminating molecules co-purifying with DNA may complicate analyses. In addition, naturally occurring modifications, as well as DNA damage occurring during sample preparation may be a challenge. One example of the latter is the strongly enhanced rate of deamination of cytosine to uracil in nucleotides and nucleosides compared with cytosine in double-stranded DNA. In spite of inherent problems, mass spectrometry has been used successfully for detection and quantification of uracil and other lesions or modifications in DNA. However, analyses of the sequence context in which such lesions occur, remains less well established.

Uracil holds a rather unique position among the non-canonical DNA bases in that it can be formed both enzymatically and spontaneously in DNA from cytosine. It can also be introduced via dUTP by DNA polymerases. Furthermore, it can be excised by DNA glycosylases to initiate DNA strand cleavage and nucleotide replacement. These characteristics provide opportunities to exploit uracil as a tool within molecular biology and medicine, but also represent inherent problems regarding DNA analysis, especially when sequencing ancient DNA. The following chapter will describe some analytical methods in which DNA-uracil constitutes an important intermediate. Finally, inherent problems associated with spontaneous cytosine deamination will be discussed in the context of analysis of stored archive samples and ancient DNA.

The following section is included:

• Index