Enhancement of NEIL1 Protein-initiated Oxidized DNA Base Excision Repair by Heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) via Direct Interaction

Repair of oxidized base lesions in the human genome, initiated by DNA glycosylases, occurs via the base excision repair pathway using conserved repair and some non-repair proteins. However, the functions of the latter noncanonical proteins in base excision repair are unclear. Here we elucidated the role of heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), identified in the immunoprecipitate of human NEIL1, a major DNA glycosylase responsible for oxidized base repair. hnRNP-U directly interacts with NEIL1 in vitro via the NEIL1 common interacting C-terminal domain, which is dispensable for its enzymatic activity. Their in-cell association increases after oxidative stress. hnRNP-U stimulates the NEIL1 in vitro base excision activity for 5-hydroxyuracil in duplex, bubble, forked, or single-stranded DNA substrate, primarily by enhancing product release. Using eluates from FLAG-NEIL1 immunoprecipitates from human cells, we observed 3-fold enhancement in complete repair activity after oxidant treatment. The lack of such enhancement in hnRNP-U-depleted cells suggests its involvement in repairing enhanced base damage after oxidative stress. The NEIL1 disordered C-terminal region binds to hnRNP-U at equimolar ratio with high affinity (K_d = ∼54 nm). The interacting regions in hnRNP-U, mapped to both termini, suggest their proximity in the native protein; these are also disordered, based on PONDR (Predictor of Naturally Disordered Regions) prediction and circular dichroism spectra. Finally, depletion of hnRNP-U and NEIL1 epistatically sensitized human cells at low oxidative genome damage, suggesting that the hnRNP-U protection of cells after oxidative stress is largely due to enhancement of NEIL1-mediated repair.     

Role of human DNA glycosylase Nei-like 2 (NEIL2) and single strand break repair protein polynucleotide kinase 3'-phosphatase in maintenance of mitochondrial genome

The repair of reactive oxygen species-induced base lesions and single strand breaks (SSBs) in the nuclear genome via the base excision (BER) and SSB repair (SSBR) pathways, respectively, is well characterize, and important for maintaining genomic integrity. However, the role of mitochondrial (mt) BER and SSBR proteins in mt genome maintenance is not completely clear. Here we show the presence of the oxidized base-specific DNA glycosylase Nei-like 2 (NEIL2) and the DNA end-processing enzyme polynucleotide kinase 3'-phosphatase (PNKP) in purified human mitochondrial extracts (MEs). Confocal microscopy revealed co-localization of PNKP and NEIL2 with the mitochondrion-specific protein cytochrome c oxidase subunit 2 (MT-CO2). Further, chromatin immunoprecipitation analysis showed association of NEIL2 and PNKP with the mitochondrial genes MT-CO2 and MT-CO3 (cytochrome c oxidase subunit 3); importantly, both enzymes also associated with the mitochondrion-specific DNA polymerase γ. In cell association of NEIL2 and PNKP with polymerase γ was further confirmed by proximity ligation assays. PNKP-depleted ME showed a significant decrease in both BER and SSBR activities, and PNKP was found to be the major 3'-phosphatase in human ME. Furthermore, individual depletion of NEIL2 and PNKP in human HEK293 cells caused increased levels of oxidized bases and SSBs in the mt genome, respectively. Taken together, these studies demonstrate the critical role of NEIL2 and PNKP in maintenance of the mammalian mitochondrial genome.     

Conserved Structural Chemistry for Incision Activity in Structurally Non-homologous Apurinic/Apyrimidinic Endonuclease APE1 and Endonuclease IV DNA Repair Enzymes

Non-coding apurinic/apyrimidinic (AP) sites in DNA form spontaneously and as DNA base excision repair intermediates are the most common toxic and mutagenic in vivo DNA lesion. For repair, AP sites must be processed by 5′ AP endonucleases in initial stages of base repair. Human APE1 and bacterial Nfo represent the two conserved 5′ AP endonuclease families in the biosphere; they both recognize AP sites and incise the phosphodiester backbone 5′ to the lesion, yet they lack similar structures and metal ion requirements. Here, we determined and analyzed crystal structures of a 2.4 Å resolution APE1-DNA product complex with Mg²⁺ and a 0.92 Å Nfo with three metal ions. Structural and biochemical comparisons of these two evolutionarily distinct enzymes characterize key APE1 catalytic residues that are potentially functionally similar to Nfo active site components, as further tested and supported by computational analyses. We observe a magnesium-water cluster in the APE1 active site, with only Glu-96 forming the direct protein coordination to the Mg²⁺. Despite differences in structure and metal requirements of APE1 and Nfo, comparison of their active site structures surprisingly reveals strong geometric conservation of the catalytic reaction, with APE1 catalytic side chains positioned analogously to Nfo metal positions, suggesting surprising functional equivalence between Nfo metal ions and APE1 residues. The finding that APE1 residues are positioned to substitute for Nfo metal ions is supported by the impact of mutations on activity. Collectively, the results illuminate the activities of residues, metal ions, and active site features for abasic site endonucleases.     

The Interaction between Polynucleotide Kinase Phosphatase and the DNA Repair Protein XRCC1 Is Critical for Repair of DNA Alkylation Damage and Stable Association at DNA Damage Sites

XRCC1 plays a key role in the repair of DNA base damage and single-strand breaks. Although it has no known enzymatic activity, XRCC1 interacts with multiple DNA repair proteins and is a subunit of distinct DNA repair protein complexes. Here we used the yeast two-hybrid genetic assay to identify mutant versions of XRCC1 that are selectively defective in interacting with a single protein partner. One XRCC1 mutant, A482T, that was defective in binding to polynucleotide kinase phosphatase (PNKP) not only retained the ability to interact with partner proteins that bind to different regions of XRCC1 but also with aprataxin and aprataxin-like factor whose binding sites overlap with that of PNKP. Disruption of the interaction between PNKP and XRCC1 did not impact their initial recruitment to localized DNA damage sites but dramatically reduced their retention there. Furthermore, the interaction between PNKP and the DNA ligase IIIα-XRCC1 complex significantly increased the efficiency of reconstituted repair reactions and was required for complementation of the DNA damage sensitivity to DNA alkylation agents of xrcc1 mutant cells. Together our results reveal novel roles for the interaction between PNKP and XRCC1 in the retention of XRCC1 at DNA damage sites and in DNA alkylation damage repair.     

A role for the base excision repair enzyme NEIL3 in replication-dependent repair of interstrand DNA cross-links derived from psoralen and abasic sites

Interstrand DNA–DNA cross-links are highly toxic lesions that are important in medicinal chemistry, toxicology, and endogenous biology. In current models of replication-dependent repair, stalling of a replication fork activates the Fanconi anemia pathway and cross-links are “unhooked” by the action of structure-specific endonucleases such as XPF-ERCC1 that make incisions flanking the cross-link. This process generates a double-strand break, which must be subsequently repaired by homologous recombination. Recent work provided evidence for a new, incision-independent unhooking mechanism involving intrusion of a base excision repair (BER) enzyme, NEIL3, into the world of cross-link repair. The evidence suggests that the glycosylase action of NEIL3 unhooks interstrand cross-links derived from an abasic site or the psoralen derivative trioxsalen. If the incision-independent NEIL3 pathway is blocked, repair reverts to the incision-dependent route. In light of the new model invoking participation of NEIL3 in cross-link repair, we consider the possibility that various BER glycosylases or other DNA-processing enzymes might participate in the unhooking of chemically diverse interstrand DNA cross-links.     

Construction and evaluation of the novel DNA vaccine harboring the inhibin α (1–32) and the RF-amide related peptide-3 genes for improving fertility in mice

To further improve fertility of animals, a novel gene RFRP-3 (RF-amide related peptide-3, RFRP-3) was used to construct DNA vaccines with INH α (1–32) (inhibin, INH) fragment for the first time. The aim of this study was to evaluate the effects of novel DNA vaccines on fertility in mice. Synthesized SINH and SRFRP (INH and RFRP genes were separately ligated to the C-terminus of the small envelope protein of the hepatitis B virus (HBV-S) gene) fragments were inserted into multiple cloning site of pIRES vector to develop p-SINH/SRFRP. The synthesized tissue plasminogen activator (TPA) signal sequence was then inserted into the p-SINH/SRFRP to construct p-TPA-SINH/TPA-SFRFP. Meanwhile, p-SINH was prepared and considered as positive control. Forty Kunming mice were equally divided into four groups and respectively immunized by electroporation with p-SINH, p-SINH/SRFRP and p-TPA-SINH/TPA-SRFRP vaccine (three times at 2 weeks interval) and saline as control. Results showed that the average antibodies (P/N value) of anti-INH and anti-RFRP in mice inoculated with p-TPA-SINH/TPA-SFRFP were significantly higher (P<0.05) than those inoculated with p-SINH/SRFRP and the positive rates were 100% (anti-INH) and 90% (anti-RFRP) respectively, at 2 weeks after the third immunization. Litter size of mice immunized with the three recombinant plasmids was higher (P<0.05) than that of the control, and litter size of mice immunized with p-TPA-SINH/TPA-SRFRP significantly increased (P<0.05) compared with p-SINH. These results suggested that the p-TPA-SINH/TPA-SRFRP harboring INH and RFRP genes was successfully constructed and had good immunogenicity, and might effectively increase litter size.     

α‐MSH tripeptide analogs activate the melanocortin 1 receptor and reduce UV‐induced DNA damage in human melanocytes

One skin cancer prevention strategy that we are developing is based on synthesizing and testing melanocortin analogs that reduce and repair DNA damage resulting from exposure to solar ultraviolet (UV) radiation, in addition to stimulating pigmentation. Previously, we reported the effects of tetrapeptide analogs of α‐melanocortin (α‐MSH) that were more potent and stable than the physiological α‐MSH, and mimicked its photoprotective effects against UV‐induced DNA damage in human melanocytes. Here, we report on a panel of tripeptide analogs consisting of a modified α‐MSH core His⁶‐d ‐Phe⁷‐Arg⁸, which contained different N‐capping groups, C‐terminal modifications, or arginine mimics. The most potent tripeptides in activating cAMP formation and tyrosinase of human melanocytes were three analogs with C‐terminal modifications. The most effective C‐terminal tripeptide mimicked α‐MSH in reducing hydrogen peroxide generation and enhancing nucleotide excision repair following UV irradiation. The effects of these three analogs required functional MC1R, as they were absent in human melanocytes that expressed non‐functional receptor. These results demonstrate activation of the MC1R by tripeptide melanocortin analogs. Designing small analogs for topical delivery should prove practical and efficacious for skin cancer prevention.     

A key role for Ctf4 in coupling the MCM2‐7 helicase to DNA polymerase α within the eukaryotic replisome

The eukaryotic replisome is a crucial determinant of genome stability, but its structure is still poorly understood. We found previously that many regulatory proteins assemble around the MCM2‐7 helicase at yeast replication forks to form the replisome progression complex (RPC), which might link MCM2‐7 to other replisome components. Here, we show that the RPC associates with DNA polymerase α that primes each Okazaki fragment during lagging strand synthesis. Our data indicate that a complex of the GINS and Ctf4 components of the RPC is crucial to couple MCM2‐7 to DNA polymerase α. Others have found recently that the Mrc1 subunit of RPCs binds DNA polymerase epsilon, which synthesises the leading strand at DNA replication forks. We show that cells lacking both Ctf4 and Mrc1 experience chronic activation of the DNA damage checkpoint during chromosome replication and do not complete the cell cycle. These findings indicate that coupling MCM2‐7 to replicative polymerases is an important feature of the regulation of chromosome replication in eukaryotes, and highlight a key role for Ctf4 in this process.     

Structural basis for the interaction between the first SURP domain of the SF3A1 subunit in U2 snRNP and the human splicing factor SF1

SURP domains are exclusively found in splicing‐related proteins in all eukaryotes. SF3A1, a component of the U2 snRNP, has two tandem SURP domains, SURP1, and SURP2. SURP2 is permanently associated with a specific short region of SF3A3 within the SF3A protein complex whereas, SURP1 binds to the splicing factor SF1 for recruitment of U2 snRNP to the early spliceosomal complex, from which SF1 is dissociated during complex conversion. Here, we determined the solution structure of the complex of SURP1 and the human SF1 fragment using nuclear magnetic resonance (NMR) methods. SURP1 adopts the canonical topology of α1–α2–3₁₀–α3, in which α1 and α2 are connected by a single glycine residue in a particular backbone conformation, allowing the two α‐helices to be fixed at an acute angle. A hydrophobic patch, which is part of the characteristic surface formed by α1 and α2, specifically contacts a hydrophobic cluster on a 16‐residue α‐helix of the SF1 fragment. Furthermore, whereas only hydrophobic interactions occurred between SURP2 and the SF3A3 fragment, several salt bridges and hydrogen bonds were found between the residues of SURP1 and the SF1 fragment. This finding was confirmed through mutational studies using bio‐layer interferometry. The study also revealed that the dissociation constant between SURP1 and the SF1 fragment peptide was approximately 20 μM, indicating a weak or transient interaction. Collectively, these results indicate that the interplay between U2 snRNP and SF1 involves a transient interaction of SURP1, and this transient interaction appears to be common to most SURP domains, except for SURP2.     

EGF and TGFα modulate structural and functional differentiation of the mammary gland from pregnant mice in vitro: Possible role of the arachidonic acid pathway

Epidermal growth factor (EGF) has been suggested to be involved in mammary gland development by mitogenic stimulation of the ductal and alveolar epithelium in virgin mice. The present studies demonstrate that also in late-pregnant mice EGF leads to proliferation of the ductal, ductular, and alveolar epithelium. The mitogenic effect is associated with structural and functional dedifferentiation of alveolar cells as revealed by analysis of morphology, expression of cytosolic and secretory proteins, and fatty acid synthesis. Using a combination of metabolic inhibitors, the dedifferentiating effect of EGF could be blocked while the mitogenic action was not influenced. This finding demonstrates that the signal transduction pathway leading to dedifferentiation and mitosis can be separated, and that the dedifferentiating effect of EGF is independent of its mitogenic properties, but is probably mediated by activation of the arachidonic acid-dependent pathways (cyclo- and lipoxygenase pathways). Release of arachidonic acid from the endogenous phospholipid pool was found to be an early response of the explants to EGF. Accordingly, arachidonic acid itself proved to be capable of inducing epithelial dedifferentiation but failed to stimulate proliferation. TGFα showed qualitatively similar effects as EGF but was generally a stronger agonist. It is suggested that EGF and TGFα also play a role in mammary gland physiology during pregnancy by final developing and maintanance of the lobulo-alveolar structure in the mammary gland and prevention of premature onset of lactation, and that this is mediated through the PLA₂-arachidonic acid signalling cascade.     

Unlike in other monocotyledonous plants such as wheat and rice, the region containing the tRNA (UCC), tRNA (UCU) and CF1 ATPase α-subunit genes has not undergone recombination in the leek chloroplast DNA

The nucleotide sequence of a 1.1 kbp BamHI fragment of the leek chloroplast DNA (Allium porrum., fam. Liliaceae) has been determined. The fragment contains the 3' part of the tRNA^Gly (UCC) gene and the tRNA^Arg (UCU) gene on the same strand, and the 3' end of the atpA gene encoding the CF₁ ATPase α-subunit which is located on the opposite strand. The gene arrangement and nucleotide sequence of this fragment are similar to those of the corresponding region in the tobacco chloroplast DNA but differ significantly from what has been observed in other monocotyledonous plants such as wheat and rice, in which the region containing these genes has undergone intensive rearrangement.     

Highly sensitive and selective spectrofluorimetric approach for the rapid determination of trace benzo[α]pyrene in drinking water and in solutions leached from disposable paper cups

Based on the excellent band narrowing and background suppressing features of second‐derivative constant‐energy synchronous spectrofluorimetry with a Δ value of 1400 cm^?1, the strong fluorescence intensity for benzo[α]pyrene (BaP) obtained in dichloromethane and the use of standard addition method, a highly sensitive and selective approach for the quantitative determination of trace amount of BaP in drinking water has been established in this study. The detection and quantification limits were 0.11 and 0.37 ng L^?1, respectively, and the recoveries obtained from spiked Milli‐Q water, bottled natural spring water, tank‐purified water and tap water at different concentrations, ranged from 86.0 to 104.0%. This method has been applied for the determination of trace BaP in solution leached from disposable paper cups. The experimental results indicated that BaP was leached from paper cups when filled with hot water, but it was not detected when cool (unheated) water was used. Copyright © 2009 John Wiley & Sons, Ltd.