Atomic structures and functional implications of the archaeal RecQ-like helicase Hjm

<p>Abstract</p> <p>Background</p> <p><it>Pyrococcus furiosus </it>Hjm (<it>Pfu</it>Hjm) is a structure-specific DNA helicase that was originally identified by <it>in vitro </it>screening for Holliday junction migration activity. It belongs to helicase superfamily 2, and shares homology with the human DNA polymerase Θ (PolΘ), HEL308, and <it>Drosophila </it>Mus308 proteins, which are involved in DNA repair. Previous biochemical and genetic analyses revealed that <it>Pfu</it>Hjm preferentially binds to fork-related Y-structured DNAs and unwinds their double-stranded regions, suggesting that this helicase is a functional counterpart of the bacterial RecQ helicase, which is essential for genome maintenance. Elucidation of the DNA unwinding and translocation mechanisms by <it>Pfu</it>Hjm will require its three-dimensional structure at atomic resolution.</p> <p>Results</p> <p>We determined the crystal structures of <it>Pfu</it>Hjm, in two apo-states and two nucleotide bound forms, at resolutions of 2.0–2.7 Å. The overall structures and the local conformations around the nucleotide binding sites are almost the same, including the side-chain conformations, irrespective of the nucleotide-binding states. The architecture of Hjm was similar to that of <it>Archaeoglobus fulgidus </it>Hel308 complexed with DNA. An Hjm-DNA complex model, constructed by fitting the five domains of Hjm onto the corresponding Hel308 domains, indicated that the interaction of Hjm with DNA is similar to that of Hel308. Notably, sulphate ions bound to Hjm lie on the putative DNA binding surfaces. Electron microscopic analysis of an Hjm-DNA complex revealed substantial flexibility of the double stranded region of DNA, presumably due to particularly weak protein-DNA interactions. Our present structures allowed reasonable homology model building of the helicase region of human PolΘ, indicating the strong conformational conservation between archaea and eukarya.</p> <p>Conclusion</p> <p>The detailed comparison between our DNA-free <it>Pfu</it>Hjm structure and the structure of Hel308 complexed with DNA suggests similar DNA unwinding and translocation mechanisms, which could be generalized to all of the members in the same family. Structural comparison also implied a minor rearrangement of the five domains during DNA unwinding reaction. The unexpected small contact between the DNA duplex region and the enzyme appears to be advantageous for processive helicase activity.</p

Suppression of TopBP1 function increases the efficacy of chemotherapeutic treatments by enhancing the induction of apoptosis

福岡歯科大学2020年

MLH1-mediated recruitment of FAN1 to chromatin for the induction of apoptosis triggered by O6-methylguanine

O6 -Methylguanines (O6 -meG), which are produced in DNA by the action of alkylating agents, are mutagenic and cytotoxic, and induce apoptosis in a mismatch repair (MMR) protein-dependent manner. To understand the molecular mechanism of O6 -meG-induced apoptosis, we performed functional analyses of FANCD2 and FANCI-associated nuclease 1 (FAN1), which was identified as an interacting partner of MLH1. Immunoprecipitation analyses showed that FAN1 interacted with both MLH1 and MSH2 after treatment with N-methyl-N-nitrosourea (MNU), indicating the formation of a FAN1-MMR complex. In comparison with control cells, FAN1-knockdown cells were more resistant to MNU, and the appearances of a sub-G1 population and caspase-9 activation were suppressed. FAN1 formed nuclear foci in an MLH1-dependent manner after MNU treatment, and some were colocalized with both MLH1 foci and single-stranded DNA (ssDNA) created at damaged sites. Under the same condition, FANCD2 also formed nuclear foci, although it was dispensable for the formation of FAN1 foci and ssDNA. MNU-induced formation of ssDNA was dramatically suppressed in FAN1-knockdown cells. We therefore propose that FAN1 is loaded on chromatin through the interaction with MLH1 and produces ssDNA by its exonuclease activity, which contributes to the activation of the DNA damage response followed by the induction of apoptosis triggered by O6 -meG.福岡歯科大

When DNA replication and protein synthesis come together.

International audienceIn all organisms, DNA and protein are synthesized by dedicated, but unrelated, machineries that move along distinct templates with no apparent coordination. Therefore, connections between DNA replication and translation are a priori unexpected. However, recent findings support the existence of such connections throughout the three domains of life. In particular, we recently identified in archaeal genomes a conserved association between genes encoding DNA replication and ribosome-related proteins which all have eukaryotic homologs. We believe that this gene organization is biologically relevant and, moreover, that it suggests the existence of a mechanism coupling DNA replication and translation in Archaea and Eukarya

The Identification of a Novel Gene, <em>MAPO2</em>, That Is Involved in the Induction of Apoptosis Triggered by O⁶-Methylguanine

<div><p>O⁶-Methylguanine, one of alkylated DNA bases, is especially mutagenic. Cells containing this lesion are eliminated by induction of apoptosis, associated with the function of mismatch repair (MMR) proteins. A retrovirus-mediated gene-trap mutagenesis was used to isolate new genes related to the induction of apoptosis, triggered by the treatment with an alkylating agent, <em>N</em>-methyl-<em>N</em>-nitrosourea (MNU). This report describes the identification of a novel gene, <em>MAPO2</em> (O⁶-<u>m</u>ethylguanine-induced <u>apo</u>ptosis <u>2</u>), which is originally annotated as <em>C1orf201</em>. The <em>MAPO2</em> gene is conserved among a wide variety of multicellular organisms and encodes a protein containing characteristic PxPxxY repeats. To elucidate the function of the gene product in the apoptosis pathway, a human cell line derived from HeLa MR cells, in which the <em>MAPO2</em> gene was stably knocked down by expressing specific miRNA, was constructed. The knockdown cells grew at the same rate as HeLa MR, thus indicating that MAPO2 played no role in the cellular growth. After exposure to MNU, HeLa MR cells and the knockdown cells underwent cell cycle arrest at G₂/M phase, however, the production of the sub-G₁ population in the knockdown cells was significantly suppressed in comparison to that in HeLa MR cells. Moreover, the activation of BAK and caspase-3, and depolarization of mitochondrial membrane, hallmarks for the induction of apoptosis, were also suppressed in the knockdown cells. These results suggest that the <em>MAPO2</em> gene product might positively contribute to the induction of apoptosis triggered by O⁶-methylguanine.</p> </div

The effect of <i>MAPO2</i> knockdown on the activation of BAK proteins.

<p>Soluble protein fractions were extracted from HeLa MR and RF101 cells harvested 2, 3, 4 and 5 days after MNU treatment. The extracts were treated with disulphide bonding inducer to form intermolecularly linked active BAK dimers. The samples were boiled with (reduced) or without (CuPhe) 2-mercaptoethanol and subjected onto SDS-PAGE followed by immunoblotting analysis to detect the total amounts of BAK monomers and active BAK dimers, respectively, using anti-BAK monoclonal antibody ab-1. β-Actin was used as a loading control. The molecular weights are shown on the left.</p

Construction of a HeLa MR-derived <i>MAPO2</i>-knockdown cell line.

<p>A) The relative expression levels of the <i>MAPO2</i> gene in HeLa MR and its derivative RF101 cells, as measured by quantitative real-time RT-PCR. (B) The growth rate of the two types of cells without MNU treatment. The numbers of cells grown under normal conditions were counted every 24 h and plotted. The experiments were performed 3 times, independently, and average values from the 3 experiments were plotted. Diamonds, HeLa MR; triangles, RF101. (C) The levels of MMR proteins. The expression levels of MMR proteins in HeLa MR and RF101 cells were analyzed using immunoblotting with anti-MSH2, anti-MSH6, anti-MLH1 and anti-PMS2 antibodies. β-Actin was used as a loading control.</p