Intraoperative Integrated Diagnostic System for Malignant Central Nervous System Tumors.

PURPOSE: The 2021 World Health Organization (WHO) classification of central nervous system (CNS) tumors uses an integrated approach involving histopathology and molecular profiling. Because majority of adult malignant brain tumors are gliomas and primary CNS lymphomas (PCNSL), rapid differentiation of these diseases is required for therapeutic decisions. In addition, diffuse gliomas require molecular information on single-nucleotide variants (SNV), such as IDH1/2. Here, we report an intraoperative integrated diagnostic (i-ID) system to classify CNS malignant tumors, which updates legacy frozen-section (FS) diagnosis through incorporation of a qPCR-based genotyping assay. EXPERIMENTAL DESIGN: FS evaluation, including GFAP and CD20 rapid IHC, was performed on adult malignant CNS tumors. PCNSL was diagnosed through positive CD20 and negative GFAP immunostaining. For suspected glioma, genotyping for IDH1/2, TERT SNV, and CDKN2A copy-number alteration was routinely performed, whereas H3F3A and BRAF SNV were assessed for selected cases. i-ID was determined on the basis of the 2021 WHO classification and compared with the permanent integrated diagnosis (p-ID) to assess its reliability. RESULTS: After retrospectively analyzing 153 cases, 101 cases were prospectively examined using the i-ID system. Assessment of IDH1/2, TERT, H3F3AK27M, BRAFV600E, and CDKN2A alterations with i-ID and permanent genomic analysis was concordant in 100%, 100%, 100%, 100%, and 96.4%, respectively. Combination with FS and intraoperative genotyping assay improved diagnostic accuracy in gliomas. Overall, i-ID matched with p-ID in 80/82 (97.6%) patients with glioma and 18/19 (94.7%) with PCNSL. CONCLUSIONS: The i-ID system provides reliable integrated diagnosis of adult malignant CNS tumors.

Sld7, an Sld3-associated protein required for efficient chromosomal DNA replication in budding yeast.

Genetic screening of yeast for sld (synthetic lethality with dpb11) mutations has identified replication proteins, including Sld2, -3, and -5, and clarified the molecular mechanisms underlying eukaryotic chromosomal DNA replication. Here, we report a new replication protein, Sld7, identified by rescreening of sld mutations. Throughout the cell cycle, Sld7 forms a complex with Sld3, which associates with replication origins in a complex with Cdc45, binds to Dpb11 when phosphorylated by cyclin-dependent kinase, and dissociates from origins once DNA replication starts. However, Sld7 does not move with the replication fork. Sld7 binds to the nonessential N-terminal portion of Sld3 and reduces its affinity for Cdc45, a component of the replication fork. Although Sld7 is not essential for cell growth, its absence reduces the level of cellular Sld3, delays the dissociation from origins of GINS, a component of the replication fork, and slows S-phase progression. These results suggest that Sld7 is required for the proper function of Sld3 at the initiation of DNA replication.

Action mechanism of human SMUG1 uracil-DNA glycosylase.

The DNA lesions resulting from deamination or oxidation of bases are generally repaired by the base excision repair pathway initiated by damage-specific DNA glycosylases. Single-strand selective monofunctional uracil-DNA glycosylase (SMUG1) present in vertebrates and insects excises not only uracil but also uracil derivatives bearing an oxidized group at ring-C5 from DNA, indicating roles in the repair of both deamination and oxidation damage to DNA. In the present study, we have constructed a series of active site mutants of human SMUG1 and analyzed the catalytic and precision damage recognition mechanisms.

Mutational analysis of the damage-recognition and catalytic mechanism of human SMUG1 DNA glycosylase.

Single-strand selective monofunctional uracil-DNA glycosylase (SMUG1), previously thought to be a backup enzyme for uracil-DNA glycosylase, has recently been shown to excise 5-hydroxyuracil (hoU), 5-hydroxymethyluracil (hmU) and 5-formyluracil (fU) bearing an oxidized group at ring C5 as well as an uracil. In the present study, we used site-directed mutagenesis to construct a series of mutants of human SMUG1 (hSMUG1), and tested their activity for uracil, hoU, hmU, fU and other bases to elucidate the catalytic and damage-recognition mechanism of hSMUG1. The functional analysis of the mutants, together with the homology modeling of the hSMUG1 structure based on that determined recently for Xenopus laevis SMUG1, revealed the crucial residues for the rupture of the N-glycosidic bond (Asn85 and His239), discrimination of pyrimidine rings through pi-pi stacking to the base (Phe98) and specific hydrogen bonds to the Watson-Crick face of the base (Asn163) and exquisite recognition of the C5 substituent through water-bridged (uracil) or direct (hoU, hmU and fU) hydrogen bonds (Gly87-Met91). Integration of the present results and the structural data elucidates how hSMUG1 accepts uracil, hoU, hmU and fU as substrates, but not other oxidized pyrimidines such as 5-hydroxycytosine, 5-formylcytosine and thymine glycol, and intact pyrimidines such as thymine and cytosine.

Identification and characterization of mammalian 5-formyluracil-DNA glycosylase.

5-Formyluracil is a major oxidative thymine lesion with mutagenic and cytotoxic properties. In this study, we have partially purified and characterized a mammalian 5-formyluracil-DNA glycosylase (FDG) from rat liver. FDG was a monofunctional DNA glycosylase and removed 5-formyluracil, uracil, 5-hydroxyuracil, 5-hydroxylmethyluracil in single-stranded and double-stranded DNA. Several lines of evidence indicate that FDG is a rat SMUG1 homologue. Human SMUG1 also exhibited similar enzymatic properties.

Repair roles of hSMUG1 assessed by damage specificity and cellular activity.

Single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1) was previously identified as a putative backup enzyme of major mammalian uracil-DNA glycosylase (UDG). However, the subsequent studies have shown conflicting results about the substrate specificity of SMUG1. In the present study, to clarify the repair role of SMUG1, we determined the damage specificity of purified human SMUG1 (hSMUG1) and its contribution to repair of oxidized bases in HeLa cell extracts.

Mammalian 5-formyluracil-DNA glycosylase. 1. Identification and characterization of a novel activity that releases 5-formyluracil from DNA.

5-Formyluracil (fU) is a major oxidative thymine lesion produced by reactive oxygen species and exhibits genotoxic and cytotoxic effects via several mechanisms. In the present study, we have searched for and characterized mammalian fU-DNA glycosylase (FDG) using two approaches. In the first approach, the FDG activity was examined using purified base excision repair enzymes. Human and mouse endonuclease III homologues (NTH1) showed a very weak FDG activity, but the parameter analysis and NaBH(4) trapping assays of the Schiff base intermediate revealed that NTH1 was kinetically incompetent for repair of fU. In the second approach, FDG was partially purified (160-fold) from rat liver. The enzyme was a monofunctional DNA glycosylase and recognized fU in single-stranded (ss) and double-stranded (ds) DNA. The most purified FDG fraction also exhibited monofunctional DNA glycosylase activities for uracil (U), 5-hydroxyuracil (hoU), and 5-hydroxymethyluracil (hmU) in ssDNA and dsDNA. The fU-excising activity of FDG was competitively inhibited by dsDNA containing U.G, hoU.G, and hmU.A but not by intact dsDNA containing T.A. Furthermore, the activities of FDG for fU, hmU, hoU, and U in ssDNA and dsDNA were neutralized by the antibody raised against SMUG1 uracil-DNA glycosylase, showing that FDG is a rat homologue of SMUG1.

Mammalian 5-formyluracil-DNA glycosylase. 2. Role of SMUG1 uracil-DNA glycosylase in repair of 5-formyluracil and other oxidized and deaminated base lesions.

In the accompanying paper [Matsubara, M., et al. (2003) Biochemistry 42, 4993-5002], we have partially purified and characterized rat 5-formyluracil (fU)-DNA glycosylase (FDG). Several lines of evidence have indicated that FDG is a rat homologue of single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1). We report here that rat and human SMUG1 (rSMUG1 and hSMUG1) expressed from the corresponding cDNAs indeed excise fU in single-stranded (ss) and double-stranded (ds) DNA. The enzymes also excised uracil (U) and uracil derivatives bearing an oxidized group at C5 [5-hydroxyuracil (hoU) and 5-hydroxymethyluracil (hmU)] in ssDNA and dsDNA but not analogous cytosine derivatives (5-hydroxycytosine and 5-formylcytosine) and other oxidized damage. The damage specificity and the salt concentration dependence of rSMUG1 (and hSMUG1) agreed well with those of FDG, confirming that FDG is rSMUG1. Consistent with the damage specificity above, hSMUG1 removed damaged bases from Fenton-oxidized calf thymus DNA, generating abasic sites. The amount of resulting abasic sites was about 10% of that generated by endonuclease III or 8-oxoguanine glycosylase in the same substrate. The HeLa cell extract and hSMUG1 exhibited a similar damage preference (hoU.G > hmU.A, fU.A), and the activities for fU, hmU, and hoU in the cell extract were effectively neutralized with hSMUG1 antibodies. These data indicate a dual role of hSMUG1 as a backup enzyme for UNG and a primary repair enzyme for a subset of oxidized pyrimidines such as fU, hmU, and hoU.