Base excision repair (BER) corrects DNA damage from oxidation, deamination and alkylation. Such base lesions cause little distortion to the DNA helix structure. BER is initiated by a DNA glycosylase that recognizes and removes the damaged base, leaving an abasic site that is further processed by short-patch repair or long-patch repair that largely uses different proteins to complete BER. At least 11 distinct mammalian DNA glycosylases are known, each recognizing a few related lesions, frequently with some overlap in specificities. Impressively, the damaged bases are rapidly identified in a vast excess of normal bases, without a supply of energy. BER protects against cancer, aging, and neurodegeneration and takes place both in nuclei and mitochondria. More recently, an important role of uracil-DNA glycosylase UNG2 in adaptive immunity was revealed. Furthermore, other DNA glycosylases may have important roles in epigenetics, thus expanding the repertoire of BER proteins.

Base Excision Repair

Chemical exchange saturation transfer (CEST) imaging is a relatively new magnetic resonance imaging contrast approach in which exogenous or endogenous compounds containing either exchangeable protons or exchangeable molecules are selectively saturated and after transfer of this saturation, detected indirectly through the water signal with enhanced sensitivity. The focus of this review is on basic magnetic resonance principles underlying CEST and similarities to and differences with conventional magnetization transfer contrast. In CEST magnetic resonance imaging, transfer of magnetization is studied in mobile compounds instead of semisolids. Similar to magnetization transfer contrast, CEST has contributions of both chemical exchange and dipolar cross-relaxation, but the latter can often be neglected if exchange is fast. Contrary to magnetization transfer contrast, CEST imaging requires sufficiently slow exchange on the magnetic resonance time scale to allow selective irradiation of the protons of interest. As a consequence, magnetic labeling is not limited to radio-frequency saturation but can be expanded with slower frequency-selective approaches such as inversion, gradient dephasing and frequency labeling. The basic theory, design criteria, and experimental issues for exchange transfer imaging are discussed. A new classification for CEST agents based on exchange type is proposed. The potential of this young field is discussed, especially with respect to in vivo application and translation to humans.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/mrm.22761

Chemical exchange saturation transfer (CEST): what is in a name and what isn't?

Current antiviral agents can control but not eliminate hepatitis B virus (HBV), because HBV establishes a stable nuclear covalently closed circular DNA (cccDNA). Interferon-α treatment can clear HBV but is limited by systemic side effects. We describe how interferon-α can induce specific degradation of the nuclear viral DNA without hepatotoxicity and propose lymphotoxin-β receptor activation as a therapeutic alternative. Interferon-α and lymphotoxin-β receptor activation up-regulated APOBEC3A and APOBEC3B cytidine deaminases, respectively, in HBV-infected cells, primary hepatocytes, and human liver needle biopsies. HBV core protein mediated the interaction with nuclear cccDNA, resulting in cytidine deamination, apurinic/apyrimidinic site formation, and finally cccDNA degradation that prevented HBV reactivation. Genomic DNA was not affected. Thus, inducing nuclear deaminases—for example, by lymphotoxin-β receptor activation—allows the development of new therapeutics that, in combination with existing antivirals, may cure hepatitis B.

https://science.sciencemag.org/content/sci/343/6176/1221.full.pdf

Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA.

Base excision repair (BER) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ROS). BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease (APE) to generate 3' OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA ligase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APE1, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3' phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and ligases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organelle targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.

/pdf/early-steps-in-the-dna-base-excision-single-strand-mh0ddm79pz.pdf

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells.

Catalyst-free reactions developed during the last decade and the latest developments in this emerging field are summarized with a focus on catalyst-free reactions in-water and on-water. Various named reactions, multi-component reactions and the synthesis of heterocyclic compounds are discussed including the use of various energy input systems such as microwave- and ultrasound irradiation, among others. Organic chemists and the practitioners of this art both in academia and industry hopefully will continue to design benign methodologies for organic synthesis in aqueous media under catalyst-free conditions by using alternative energy inputs based on fundamental principles.

Benign by design: catalyst-free in-water, on-water green chemical methodologies in organic synthesis

The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extrahelical base recognition mechanism. Efficient removal of uracil is essential for prevention of C-to-T transition mutations arising from cytosine deamination, cytotoxic U•A pairs arising from incorporation of dUTP in DNA, and for increasing immunoglobulin gene diversity during the acquired immune response. A central event in all of these UNG-mediated processes is the singling out of rare U•A or U•G base pairs in a background of approximately 109 T•A or C•G base pairs in the human genome. Here we establish for the human and Escherichia coli enzymes that discrimination of thymine and uracil is initiated by thermally induced opening of T•A and U•A base pairs and not by active participation of the enzyme. Thus, base-pair dynamics has a critical role in the genome-wide search for uracil, and may be involved in initial damage recognition by other DNA repair glycosylases. Uracil (U) belongs in RNA, where it takes the place filled by thymine in DNA. But if uracil appears in DNA in error, it can lead to potentially life-threatening mutations. This can typically occur by chemical modification of cytosine. To counter this threat, cells use the enzyme uracil DNA glycosylase to remove uracil from DNA. The detailed mechanism by which this enzyme polices DNA for stray uracils is now revealed. The DNA helix is not static but in a process rather like molecular 'breathing'; base pairs separate briefly then reform. When a uracil base pops out of the helix it is grabbed by uracil DNA glycosylase and removed. Thymine, differing only in one methyl group from uracil, is similarly grabbed but as it does not quite fit the policing enzyme's active site, it is released to go about its business in the DNA molecule. The enzyme uracil DNA glycosylase does not actively extrude just the uracil base from the DNA helix to facilitate its removal; instead, transient, passive opening of thymine: adenine and uracil: adenine base pairs allows both thymine and uracil to become extrahelical, but only uracil can subsequently fit in the active site.

Enzymatic capture of an extrahelical thymine in the search for uracil in DNA

A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly ordered primary sequences of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we call the search complex (SC). Sliding is frequently punctuated by the formation of a transient "interrogation" complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location.

Detection of damaged DNA bases by DNA glycosylase enzymes.

We report an “exchange-rate-filtered” magnetic resonance approach that allows the detection of exchangeable protons of low-concentration solutes without interference from nonexchanging protons. Thi...

/pdf/indirect-detection-of-labile-solute-proton-spectra-via-the-1szaeapsdp.pdf

Indirect Detection of Labile Solute Proton Spectra via the Water Signal Using Frequency-Labeled Exchange (FLEX) Transfer

Fiber optics applied to transillumination of teeth and other oral structures is a useful technic for detection of caries, calculus, and soft tissue lesions. It permits a cold, high-intensity light source to be used anywhere in the oral cavity with ease and flexibility. It often shows the degree of undermined carious tooth structure more accurately than the usual bitewing radiograph, and may aid in diagnosis of periapical and periodontal abscesses before bony destruction has occurred or swelling is apparent.

Transillumination of the Oral Cavity with Use of Fiber Optics

The DNA repair enzyme human uracil DNA glycosylase (UNG) scans short stretches of genomic DNA and captures rare uracil bases as they transiently emerge from the DNA duplex via spontaneous base pair breathing motions. The process of DNA scanning requires that the enzyme transiently loosen its grip on DNA to allow stochastic movement along the DNA contour, while engaging extrahelical bases requires motions on a more rapid timescale. Here, we use NMR dynamic measurements to show that free UNG has no intrinsic dynamic properties in the millisecond to microsecond and subnanosecond time regimes, and that the act of binding to nontarget DNA reshapes the dynamic landscape to allow productive millisecond motions for scanning and damage recognition. These results suggest that DNA structure and the spontaneous dynamics of base pairs may drive the evolution of a protein sequence that is tuned to respond to this dynamic regime.

Joshua I. Friedman

Papers

Enzymatic capture of an extrahelical thymine in the search for uracil in DNA

Detection of damaged DNA bases by DNA glycosylase enzymes.

Indirect Detection of Labile Solute Proton Spectra via the Water Signal Using Frequency-Labeled Exchange (FLEX) Transfer

Transillumination of the Oral Cavity with Use of Fiber Optics

Nontarget DNA binding shapes the dynamic landscape for enzymatic recognition of DNA damage