Hilde Nilsen

Immunoglobulin Isotype Switching Is Inhibited and Somatic Hypermutation Perturbed in UNG-Deficient Mice

BACKGROUND We have previously proposed that deamination of cytosine to uracil at sites within the immunoglobulin loci by activation-induced deaminase (AID) triggers antibody diversification. The pattern of diversification (phase 1 or 2 hypermutation, gene conversion, or switch recombination) is viewed as depending on the mode of resolution of the dU/dG lesion. A major resolution mode involves excising the uracil, an activity that at least four different enzymes can accomplish in the mouse. RESULTS Deficiency in UNG uracil-DNA glycosylase alone is sufficient to distort the pathway of hypermutation in mice. In ung(-/-) animals, mutations at dC/dG pairs are dramatically shifted toward transitions (95%), indicating that the generation of abasic sites (which can induce transversions) has been inhibited. The pattern of substitutions at dA/dT pairs is unaffected. Class-switch recombination is substantially, but not totally, inhibited. CONCLUSIONS The results provide strong support for the DNA deamination model for antibody diversification with respect to class-switching as well as hypermutation and, in the context of this model, suggest that (i) UNG is the major mouse DNA glycosylase responsible for processing the programmed dU/dG lesions within the immunoglobulin locus; (ii) the second (dA/dT-biased) phase of mutation is probably triggered by recognition of the initiating dU/dG lesion; and (iii) switch recombination largely proceeds via formation of an abasic site, although (iv) an UNG-independent pathway of switch recombination exists, which could reflect action by another uracil-DNA glycosylase but might alternatively be explained by a distinct pathway of resolution, for example, one involving MSH2/MSH6 recognition of the dU/dG lesion.

Base excision repair of DNA in mammalian cells

Base excision repair (BER) of DNA corrects a number of spontaneous and environmentally induced genotoxic or miscoding base lesions in a process initiated by DNA glycosylases. An AP endonuclease cleaves at the 5′ side of the abasic site and the repair process is subsequently completed via either short patch repair or long patch repair, which largely require different proteins. As one example, the UNG gene encodes both nuclear (UNG2) and mitochondrial (UNG1) uracil DNA glycosylase and prevents accumulation of uracil in the genome. BER is likely to have a major role in preserving the integrity of DNA during evolution and may prevent cancer.

Uracil-DNA Glycosylase (UNG)-Deficient Mice Reveal a Primary Role of the Enzyme during DNA Replication

Gene-targeted knockout mice have been generated lacking the major uracil-DNA glycosylase, UNG. In contrast to ung- mutants of bacteria and yeast, such mice do not exhibit a greatly increased spontaneous mutation frequency. However, there is only slow removal of uracil from misincorporated dUMP in isolated ung-/- nuclei and an elevated steady-state level of uracil in DNA in dividing ung-/- cells. A backup uracil-excising activity in tissue extracts from ung null mice, with properties indistinguishable from the mammalian SMUG1 DNA glycosylase, may account for the repair of premutagenic U:G mispairs resulting from cytosine deamination in vivo. The nuclear UNG protein has apparently evolved a specialized role in mammalian cells counteracting U:A base pairs formed by use of dUTP during DNA synthesis.

hUNG2 Is the Major Repair Enzyme for Removal of Uracil from U:A Matches, U:G Mismatches, and U in Single-stranded DNA, with hSMUG1 as a Broad Specificity Backup

hUNG2 and hSMUG1 are the only known glycosylases that may remove uracil from both double- and single-stranded DNA in nuclear chromatin, but their relative contribution to base excision repair remains elusive. The present study demonstrates that both enzymes are strongly stimulated by physiological concentrations of Mg2+ , at which the activity of hUNG2 is 2–3 orders of magnitude higher than of hSMUG1. Moreover, Mg2+ increases the preference of hUNG2 toward uracil in ssDNA nearly 40-fold. APE1 has a strong stimulatory effect on hSMUG1 against dsU, apparently because of enhanced dissociation of hSMUG1 from AP sites in dsDNA. hSMUG1 also has a broader substrate specificity than hUNG2, including 5-hydroxymethyluracil and 3,N 4-ethenocytosine. hUNG2 is excluded from, whereas hSMUG1 accumulates in, nucleoli in living cells. In contrast, only hUNG2 accumulates in replication foci in the S-phase. hUNG2 in nuclear extracts initiates base excision repair of plasmids containing either U:A and U:G in vitro. Moreover, an additional but delayed repair of the U:G plasmid is observed that is not inhibited by neutralizing antibodies against hUNG2 or hSMUG1. We propose a model in which hUNG2 is responsible for both prereplicative removal of deaminated cytosine and postreplicative removal of misincorporated uracil at the replication fork. We also provide evidence that hUNG2 is the major enzyme for removal of deaminated cytosine outside of replication foci, with hSMUG1 acting as a broad specificity backup.

Defective Mitophagy in XPA via PARP-1 Hyperactivation and NAD+/SIRT1 Reduction

Mitochondrial dysfunction is a common feature in neurodegeneration and aging. We identify mitochondrial dysfunction in xeroderma pigmentosum group A (XPA), a nucleotide excision DNA repair disorder with severe neurodegeneration, in silico and in vivo. XPA-deficient cells show defective mitophagy with excessive cleavage of PINK1 and increased mitochondrial membrane potential. The mitochondrial abnormalities appear to be caused by decreased activation of the NAD(+)-SIRT1-PGC-1α axis triggered by hyperactivation of the DNA damage sensor PARP-1. This phenotype is rescued by PARP-1 inhibition or by supplementation with NAD(+) precursors that also rescue the lifespan defect in xpa-1 nematodes. Importantly, this pathogenesis appears common to ataxia-telangiectasia and Cockayne syndrome, two other DNA repair disorders with neurodegeneration, but absent in XPC, a DNA repair disorder without neurodegeneration. Our findings reveal a nuclear-mitochondrial crosstalk that is critical for the maintenance of mitochondrial health.

Excision of deaminated cytosine from the vertebrate genome: role of the SMUG1 uracil–DNA glycosylase

Gene‐targeted mice deficient in the evolutionarily conserved uracil–DNA glycosylase encoded by the UNG gene surprisingly lack the mutator phenotype characteristic of bacterial and yeast ung− mutants. A complementary uracil–DNA glycosylase activity detected in ung−/− murine cells and tissues may be responsible for the repair of deaminated cytosine residues in vivo. Here, specific neutralizing antibodies were used to identify the SMUG1 enzyme as the major uracil–DNA glycosylase in UNG‐deficient mice. SMUG1 is present at similar levels in cell nuclei of non‐proliferating and proliferating tissues, indicating a replication‐ independent role in DNA repair. The SMUG1 enzyme is found in vertebrates and insects, whereas it is absent in nematodes, plants and fungi. We propose a model in which SMUG1 has evolved in higher eukaryotes as an anti‐mutator distinct from the UNG enzyme, the latter being largely localized to replication foci in mammalian cells to counteract de novo dUMP incorporation into DNA.

Gene-targeted mice lacking the Ung uracil-DNA glycosylase develop B-cell lymphomas

Mice deficient in the Ung uracil-DNA glycosylase have an increased level of uracil in their genome, consistent with a major role of Ung counteracting U : A base pairs arising by misincorporation of dUMP during DNA replication. A complementary uracil-excising activity apparently acts on premutagenic U : G lesions resulting from deamination of cytosine throughout the genome. However, Ung specifically processes U : G lesions targeted to immunoglobulin variable (V) genes during somatic hypermutation and class-switch recombination. Gene-targeted Ung−/− null mice remained tumour-free and showed no overt pathological phenotype up to ∼12 months of age. We have monitored a large cohort of ageing Ung−/− mice and, beyond 18 months of age, they had a higher morbidity than Ung+/+ controls. Post-mortem analyses revealed pathological changes in lymphoid organs, abnormal lymphoproliferation, and a greatly increased incidence of B-cell lymphomas in older Ung-deficient mice. These are the first data reporting the development of spontaneous malignancies in mice due to deficiency in a DNA glycosylase. Furthermore, they support a specific role for Ung in the immune system, with lymphomagenesis being related to perturbed processing of antibody genes in germinal centre B cells.

DNA base excision repair of uracil residues in reconstituted nucleosome core particles

The human base excision repair machinery must locate and repair DNA base damage present in chromatin, of which the nucleosome core particle is the basic repeating unit. Here, we have utilized fragments of the Lytechinus variegatus 5S rRNA gene containing site‐specific U:A base pairs to investigate the base excision repair pathway in reconstituted nucleosome core particles in vitro. The human uracil‐DNA glycosylases, UNG2 and SMUG1, were able to remove uracil from nucleosomes. Efficiency of uracil excision from nucleosomes was reduced 3‐ to 9‐fold when compared with naked DNA, and was essentially uniform along the length of the DNA substrate irrespective of rotational position on the core particle. Furthermore, we demonstrate that the excision repair pathway of an abasic site can be reconstituted on core particles using the known repair enzymes, AP‐endonuclease 1, DNA polymerase β and DNA ligase III. Thus, base excision repair can proceed in nucleosome core particles in vitro, but the repair efficiency is limited by the reduced activity of the uracil‐DNA glycosylases and DNA polymerase β on nucleosome cores.

Properties and functions of human uracil-DNA glycosylase from the UNG gene.

The human UNG-gene at position 12q24.1 encodes nuclear (UNG2) and mitochondrial (UNG1) forms of uracil-DNA glycosylase using differentially regulated promoters, PA and PB, and alternative splicing to produce two proteins with unique N-terminal sorting sequences. PCNA and RPA co-localize with UNG2 in replication foci and interact with N-terminal sequences in UNG2. Mitochondrial UNG1 is processed to shorter forms by mitochondrial processing peptidase (MPP) and an unidentified mitochondrial protease. The common core catalytic domain in UNG1 and UNG2 contains a conserved DNA binding groove and a tight-fitting uracil-binding pocket that binds uracil only when the uracil-containing nucleotide is flipped out. Certain single amino acid substitutions in the active site of the enzyme generate DNA glycosylases that remove either thymine or cytosine. These enzymes induce cytotoxic and mutagenic abasic (AP) sites in the E. coli chromosome and were used to examine biological consequences of AP sites. It has been assumed that a major role of the UNG gene product(s) is to repair mutagenic U:G mispairs caused by cytosine deamination. However, one major role of UNG2 is to remove misincorporated dUMP residues. Thus, knockout mice deficient in Ung activity (Ung-/- mice) have only small increases in GC-->AT transition mutations, but Ung-/- cells are deficient in removal of misincorporated dUMP and accumulate approximately 2000 uracil residues per cell. We propose that BER is important both in the prevention of cancer and for preserving the integrity of germ cell DNA during evolution.

Abrogation of the CLK‐2 checkpoint leads to tolerance to base‐excision repair intermediates

Incorporation of uracil during DNA synthesis is among the most common types of endogenously generated DNA damage. Depletion of Caenorhabditis elegans dUTPase by RNA interference allowed us to study the role of DNA damage response (DDR) pathways when responding to high levels of uracil in DNA. dUTPase depletion compromised development, caused embryonic lethality and led to activation of cell‐cycle arrest and apoptosis. These phenotypes manifested as a result of processing misincorporated uracil by the uracil‐DNA glycosylase UNG‐1. Strikingly, abrogation of the clk‐2 checkpoint gene rescued lethality and developmental defects, and eliminated cell‐cycle arrest and apoptosis after dUTPase depletion. These data show a genetic interaction between UNG‐1 and activation of the CLK‐2 DDR pathway after uracil incorporation into DNA. Our results indicate that persistent repair intermediates and/or single‐stranded DNA formed during repair of misincorporated uracil are tolerated in the absence of the CLK‐2 checkpoint in C. elegans.