Lars Hagen

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.

Morphine glucuronide-to-morphine plasma ratios are unaffected by the UGT2B7 H268Y and UGT1A1*28 polymorphisms in cancer patients on chronic morphine therapy

HeadingAbstractObjective. UDP-glucuronosyltransferase (UGT) 2B7 is the major UGT isoform responsible for the 3- and 6-glucuronidation of morphine in humans. Studies in rats have indicated that UGT1A1 may also contribute to the formation of morphine 3-glucuronide (M3G). Our objective was to investigate whether the UGT2B7 H268Y and UGT1A1*28 polymorphisms contribute to the variability in morphine glucuronide-to-morphine plasma ratios among cancer patients undergoing analgesic therapy with morphine.Methods. Seventy patients with normal hepatic and renal function using slow-release morphine to relieve cancer pain were included. UGT2B7 genotyping was performed using restriction enzyme analysis of polymerase chain reaction (PCR)-amplified DNA fragments. Wild-type and variant alleles of the UGT1A1 gene were identified using sizing of PCR-amplified fragments. Morphine 6-glucuronide (M6G)/morphine, M3G/morphine, and M3G/M6G plasma ratios were compared between genotypes.Results. The M3G/morphine, M6G/morphine, and M3G/M6G plasma ratios varied 16-, 42-, and sevenfold, respectively, among individuals. No statistically significant differences in plasma ratios were found between individuals possessing UGT2B7 H/H (n=20), H/Y (n=30), or Y/Y (n=20) genotypes. Five patients were homozygous for the UGT1A1 TA7 allele, which is associated with reduced UGT1A1 gene expression. However, the mean M3G/M6G and M3G/morphine plasma ratios in TA7 homozygous subjects did not differ significantly from those of heterozygous or homozygous wild-type (TA6) individuals.Conclusion. The UGT2B7 H268Y polymorphism cannot account for the considerable variation in glucuronide-to-morphine ratios in cancer patients. Moreover, the contribution of UGT1A1 to the formation of M3G appears to be of minor biological significance, at least in a UGT2B7 background.

Cell cycle‐specific UNG2 phosphorylations regulate protein turnover, activity and association with RPA

Human UNG2 is a multifunctional glycosylase that removes uracil near replication forks and in non‐replicating DNA, and is important for affinity maturation of antibodies in B cells. How these diverse functions are regulated remains obscure. Here, we report three new phosphoforms of the non‐catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin‐dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c‐myc‐like phosphodegron. The enhanced catalytic turnover of UNG2 p‐S23 likely optimises the protein to excise uracil along with rapidly moving replication forks. Our findings may aid further studies of how UNG2 initiates mutagenic rather than repair processing of activation‐induced deaminase‐generated uracil at Ig loci in B cells.

Human AlkB Homolog 1 Is a Mitochondrial Protein That Demethylates 3-Methylcytosine in DNA and RNA

The Escherichia coli AlkB protein and human homologs hABH2 and hABH3 are 2-oxoglutarate (2OG)/Fe(II)-dependent DNA/RNA demethylases that repair 1-methyladenine and 3-methylcytosine residues. Surprisingly, hABH1, which displays the strongest homology to AlkB, failed to show repair activity in two independent studies. Here, we show that hABH1 is a mitochondrial protein, as demonstrated using fluorescent fusion protein expression, immunocytochemistry, and Western blot analysis. A fraction is apparently nuclear and this fraction increases strongly if the fluorescent tag is placed at the N-terminal end of the protein, thus interfering with mitochondrial targeting. Molecular modeling of hABH1 based upon the sequence and known structures of AlkB and hABH3 suggested an active site almost identical to these enzymes. hABH1 decarboxylates 2OG in the absence of a prime substrate, and the activity is stimulated by methylated nucleotides. Employing three different methods we demonstrate that hABH1 demethylates 3-methylcytosine in single-stranded DNA and RNA in vitro. Site-specific mutagenesis confirmed that the putative Fe(II) and 2OG binding residues are essential for activity. In conclusion, hABH1 is a functional mitochondrial AlkB homolog that repairs 3-methylcytosine in single-stranded DNA and RNA.

Uracil in DNA and its processing by different DNA glycosylases.

Uracil in DNA may result from incorporation of dUMP during replication and from spontaneous or enzymatic deamination of cytosine, resulting in U:A pairs or U:G mismatches, respectively. Uracil generated by activation-induced cytosine deaminase (AID) in B cells is a normal intermediate in adaptive immunity. Five mammalian uracil-DNA glycosylases have been identified; these are mitochondrial UNG1 and nuclear UNG2, both encoded by the UNG gene, and the nuclear proteins SMUG1, TDG and MBD4. Nuclear UNG2 is apparently the sole contributor to the post-replicative repair of U:A lesions and to the removal of uracil from U:G contexts in immunoglobulin genes as part of somatic hypermutation and class-switch recombination processes in adaptive immunity. All uracil-DNA glycosylases apparently contribute to U:G repair in other cells, but they are likely to have different relative significance in proliferating and non-proliferating cells, and in different phases of the cell cycle. There are also some indications that there may be species differences in the function of the uracil-DNA glycosylases.

The rate of base excision repair of uracil is controlled by the initiating glycosylase

Uracil in DNA is repaired by base excision repair (BER) initiated by a DNA glycosylase, followed by strand incision, trimming of ends, gap filling and ligation. Uracil in DNA comes in two distinct forms; U:A pairs, typically resulting from replication errors, and mutagenic U:G mismatches, arising from cytosine deamination. To identify proteins critical to the rate of repair of these lesions, we quantified overall repair of U:A pairs, U:G mismatches and repair intermediates (abasic sites and nicked abasic sites) in vitro. For this purpose we used circular DNA substrates and nuclear extracts of eight human cell lines with wide variation in the content of BER proteins. We identified the initiating uracil-DNA glycosylase UNG2 as the major overall rate-limiting factor. UNG2 is apparently the sole glycosylase initiating BER of U:A pairs and generally initiated repair of almost 90% of the U:G mismatches. Surprisingly, TDG contributed at least as much as single-strand selective monofunctional uracil-DNA glycosylase 1 (SMUG1) to BER of U:G mismatches. Furthermore, in a cell line that expressed unusually high amounts of TDG, this glycosylase contributed to initiation of as much as approximately 30% of U:G repair. Repair of U:G mismatches was generally faster than that of U:A pairs, which agrees with the known substrate preference of UNG-type glycosylases. Unexpectedly, repair of abasic sites opposite G was also generally faster than when opposite A, and this could not be explained by the properties of the purified APE1 protein. It may rather reflect differences in substrate recognition or repair by different complex(es). Lig III is apparently a minor rate-regulator for U:G repair. APE1, Pol beta, Pol delta, PCNA, XRCC1 and Lig I did not seem to be rate-limiting for overall repair of any of the substrates. These results identify damaged base removal as the major rate-limiting step in BER of uracil in human cells.

Hexaene Derivatives of Nystatin Produced as a Result of an Induced Rearrangement within the nysC Polyketide Synthase Gene in S. noursei ATCC 11455

Genetic manipulation of the polyketide synthase (PKS) gene nysC involved in the biosynthesis of the tetraene antifungal antibiotic nystatin yielded a recombinant strain producing hexaene nystatin derivatives. Analysis of one such compound, S48HX, by LC-MS/MS suggested that it comprises a 36-membered macrolactone ring completely decorated by the post-PKS modification enzymes. Further characterization by bioassay has shown that S48HX exhibits antifungal activity. Genetic analysis of the hexaene-producing mutant revealed an in-frame deletion within the nysC gene via recombination between two homologous ketoreductase domain-encoding sequences. Apparently, this event resulted in the elimination of one complete module from NysC PKS, subsequently leading to the production of the nystatin derivative with a contracted macrolactone ring. These results represent the first example of manipulation of a PKS gene for the biosynthesis of a polyene antibiotic.

Structural characteristics of a novel antifreeze protein from the longhorn beetle Rhagium inquisitor.

Antifreeze proteins (AFPs) are characterized by their capacity to inhibit the growth of ice and are produced by a variety of polar fish, terrestrial arthropods and other organisms inhabiting cold environments. This capacity reflects their role as stabilizers of supercooled body fluids. The longhorn beetle Rhagium inquisitor is known to express AFPs in its body fluids. In this work we report on the primary structure and structural characteristics of a 12.8 kDa AFP from this beetle (RiAFP). It has a high capacity to evoke antifreeze activity as compared to other known insect AFPs and it is structurally unique in several aspects. In contrast to the high content of disulfide bond-formation observed in other coleopteran AFPs, RiAFP contains only a single such bond. Six internal repeat segments of a thirteen residue repeat pattern is irregularly spaced apart throughout its sequence. The central part of these repeat segments is preserved as TxTxTxT, which is effectively an expansion of the TxT ice-binding motif found in the AFPs of several known insect AFPs.

Randomized, double‐blind, placebo‐controlled study of the effect of rectal paracetamol on morphine consumption after abdominal hysterectomy

Background: Paracetamol is widely used for postoperative analgesia. The effect is well documented in minor and moderate extensive surgery, but the effect of paracetamol as an adjunct to opioids in major abdominal surgery is less examined.

Identification of a novel in vivo virus-targeted phosphorylation site in interferon regulatory factor-3 (IRF3).

The transcription factor interferon regulatory factor-3 (IRF3) regulates expression of type I interferon-β and plays an important role in antiviral immunity. Despite the biological importance of IRF3, its in vivo phosphorylation pattern has not been reported. In this study, we have identified residues in IRF3 that are phosphorylated in vivo after infection with Sendai virus. We found that Sendai virus induced phosphorylation of the C-terminal residues Thr390 and Ser396, in addition to either Ser385 or Ser386. Moreover, Ser173 and Ser175 were constitutively phosphorylated. Ser396 has previously been suggested to be the major target of the IRF3-activating kinase TBK1 (TANK-binding kinase-1), whereas Thr390 has not previously been implicated in IRF3 regulation. Mutagenesis studies indicated that phosphorylation of Thr390 promotes Ser396 phosphorylation and binding to the coactivator cAMP-response element-binding protein. Taken together, our results show that IRF3 is subject to multiple interdependent phosphorylations, and we identify Thr390 as a novel in vivo phosphorylation site that modulates the phosphorylation status of TBK1-targeted Ser396.