SUMMARY Flap endonuclease (FEN1), essential for DNA replication and repair, removes RNA and DNA 5′-flaps. FEN1 5′ nuclease superfamily members acting in nucleotide excision repair (XPG), mismatch repair (EXO1) and homologous recombination (GEN1) paradoxically incise structurally distinct bubbles, ends or Holliday junctions, respectively. Here structural and functional analyses of human FEN1:DNA complexes show structure-specific, sequence-independent recognition for nicked dsDNA bent 100° with unpaired 3′- and 5′-flaps. Above the active site, a helical cap over a gateway formed by two helices enforces ssDNA threading and specificity for free 5′-ends. Crystallographic analyses of product and substrate complexes reveal that dsDNA binding and bending, the ssDNA gateway, and double-base unpairing flanking the scissile phosphate control precise flap incision by the two-metal-ion active site. Superfamily conserved motifs bind and open dsDNA, direct the target region into the helical gateway permitting only non-base-paired oligonucleotides active site access, and support a unified understanding of superfamily substrate specificity.

DNA double-strand breaks (DSBs) leading to loss of nucleotides in the transcribed region can be lethal. Classical non-homologous end-joining (C-NHEJ) is the dominant pathway for DSB repair (DSBR) in adult mammalian cells. Here we report that during such DSBR, mammalian C-NHEJ proteins form a multiprotein complex with RNA polymerase II and preferentially associate with the transcribed genes after DSB induction. Depletion of C-NHEJ factors significantly abrogates DSBR in transcribed but not in non-transcribed genes. We hypothesized that nascent RNA can serve as a template for restoring the missing sequences, thus allowing error-free DSBR. We indeed found pre-mRNA in the C-NHEJ complex. Finally, when a DSB-containing plasmid with several nucleotides deleted within the E. coli lacZ gene was allowed time to repair in lacZ-expressing mammalian cells, a functional lacZ plasmid could be recovered from control but not C-NHEJ factor-depleted cells, providing important mechanistic insights into C-NHEJ-mediated error-free DSBR of the transcribed genome.

Loss of a nonenzymatic function of XPG results in defective transcription-coupled repair (TCR), Cockayne syndrome (CS), and early death, but the molecular basis for these phenotypes is unknown. Mutation of CSB, CSA, or the TFIIH helicases XPB and XPD can also cause defective TCR and CS. We show that XPG interacts with elongating RNA polymerase II (RNAPII) in the cell and binds stalled RNAPII ternary complexes in vitro both independently and cooperatively with CSB. XPG binds transcription-sized DNA bubbles through two domains not required for incision and functionally interacts with CSB on these bubbles to stimulate its ATPase activity. Bound RNAPII blocks bubble incision by XPG, but an ATP hydrolysis-dependent process involving TFIIH creates access to the junction, allowing incision. Together, these results implicate coordinated recognition of stalled transcription by XPG and CSB in TCR initiation and suggest that TFIIH-dependent remodeling of stalled RNAPII without release may be sufficient to allow repair.

Exposure to thirdhand smoke (THS) is a newly described health risk. Evidence supports its widespread presence in indoor environments. However, its genotoxic potential, a critical aspect in risk assessment, is virtually untested. An important characteristic of THS is its ability to undergo chemical transformations during aging periods, as demonstrated in a recent study showing that sorbed nicotine reacts with the indoor pollutant nitrous acid (HONO) to form tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-4-(3-pyridyl)butanal (NNA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The goal of this study was to assess the genotoxicity of THS in human cell lines using two in vitro assays. THS was generated in laboratory systems that simulated short (acute)- and long (chronic)-term exposures. Analysis by liquid chromatography-tandem mass spectrometry quantified TSNAs and common tobacco alkaloids in extracts of THS that had sorbed onto cellulose substrates. Exposure of human HepG2 cells to either acute or chronic THS for 24h resulted in significant increases in DNA strand breaks in the alkaline Comet assay. Cell cultures exposed to NNA alone showed significantly higher levels of DNA damage in the same assay. NNA is absent in freshly emitted secondhand smoke, but it is the main TSNA formed in THS when nicotine reacts with HONO long after smoking takes place. The long amplicon-quantitative PCR assay quantified significantly higher levels of oxidative DNA damage in hypoxanthine phosphoribosyltransferase 1 (HPRT) and polymerase β (POLB) genes of cultured human cells exposed to chronic THS for 24h compared with untreated cells, suggesting that THS exposure is related to increased oxidative stress and could be an important contributing factor in THS-mediated toxicity. The findings of this study demonstrate for the first time that exposure to THS is genotoxic in human cell lines.

The human endonuclease III (hNTH1), a homolog of the Escherichia coli enzyme (Nth), is a DNA glycosylase with abasic (apurinic/apyrimidinic (AP)) lyase activity and specifically cleaves oxidatively damaged pyrimidines in DNA. Its cDNA was cloned, and the full-length enzyme (304 amino acid residues) was expressed as a glutathione S-transferase fusion polypeptide in E. coli. Purified wild-type protein with two additional amino acid residues and a truncated protein with deletion of 22 residues at the NH 2 terminus were equally active and had absorbance maxima at 280 and 410 nm, the latter due to the presence of a [4Fe-4S]cluster, as in E. coli Nth. The enzyme cleaved thymine glycol-containing form I plasmid DNA and a dihydrouracil (DHU)-containing oligonucleotide duplex. The protein had a molar extinction coefficient of 5.0 ؋ 10 4 and a pI of 10. With the DHU-containing oligonucleotide duplex as substrate, the K m was 47 nM, and k cat was ϳ0.6/min, independent of whether DHU paired with G or A. The enzyme carries out ␤-elimination and forms a Schiff base between the active site residue and the deoxyribose generated after base removal. The prediction of Lys-212 being the active site was confirmed by sequence analysis of the peptideoligonucleotide adduct. Furthermore, replacing Lys-212 with Gln inactivated the enzyme. However, replacement with Arg-212 yielded an active enzyme with about 85-fold lower catalytic specificity than the wild-type protein. DNase I footprinting with hNTH1 showed protection of 10 nucleotides centered around the base lesion in the damaged strand and a stretch of 15 nucleotides (with the G opposite the lesion at the 5-boundary) in the complementary strand. Immunological studies showed that HeLa cells contain a single hNTH species of the predicted size, localized in both the nucleus and the cytoplasm.Reactive oxygen species are generated as by-products of oxidative phosphorylation or by ionizing radiation and induce extensive base damage that is mainly repaired via the base excision repair (BER) 1 pathway. This repair is initiated by removal of the damaged base, catalyzed by a DNA glycosylase (1-3). There are two classes of DNA glycosylases with distinct substrate specificities: the monofunctional simple glycosylase and the glycosylase with associated AP lyase activity. All oxidized base lesions are removed from DNA by DNA glycosylase/AP lyases, which not only catalyze removal of the base lesion but also cause strand cleavage via ␤-elimination. The Escherichia coli endonuclease III (Nth) recognizes a wide range of oxidized pyrimidine derivatives, including ring-saturated and ring-fragmented derivatives such as thymine glycol (Tg), 5-hydroxycytosine, 5,6-dihydrouracil (DHU), and urea (1, 3-6). This enzyme has been well conserved from E. coli to the humans (7,8). On the other hand, oxidized purine lesions are also repaired by DNA glycosylase/AP lyases, i.e. Mut M (Fpg) of E. coli or OGG of eukaryotes (yeast and mammals), which do not share extensive sequence similarity. Furthermore, unlike these e...