Contribution of transcription-coupled DNA repair to MMS-induced mutagenesis in E. coli strains deficient in functional AlkB protein.

In Escherichia coli the alkylating agent methyl methanesulfonate (MMS) induces defense systems (adaptive and SOS responses), DNA repair pathways, and mutagenesis. We have previously found that AlkB protein induced as part of the adaptive (Ada) response protects cells from the genotoxic and mutagenic activity of MMS. AlkB is a non-heme iron (II), alpha-ketoglutarate-dependent dioxygenase that oxidatively demethylates 1meA and 3meC lesions in DNA, with recovery of A and C. Here, we studied the impact of transcription-coupled DNA repair (TCR) on MMS-induced mutagenesis in E. coli strain deficient in functional AlkB protein. Measuring the decline in the frequency of MMS-induced argE3-->Arg(+) revertants under transient amino acid starvation (conditions for TCR induction), we have found a less effective TCR in the BS87 (alkB(-)) strain in comparison with the AB1157 (alkB(+)) counterpart. Mutation in the mfd gene encoding the transcription-repair coupling factor Mfd, resulted in weaker TCR in MMS-treated and starved AB1157 mfd-1 cells in comparison to AB1157 mfd(+), and no repair in BS87 mfd(-) cells. Determination of specificity of Arg(+) revertants allowed to conclude that MMS-induced 1meA and 3meC lesions, unrepaired in bacteria deficient in AlkB, are the source of mutations. These include AT-->TA transversions by supL suppressor formation (1meA) and GC-->AT transitions by supB or supE(oc) formation (3meC). The repair of these lesions is partly Mfd-dependent in the AB1157 mfd-1 and totally Mfd-dependent in the BS87 mfd-1 strain. The nucleotide sequence of the mfd-1 allele shows that the mutated Mfd-1 protein, deprived of the C-terminal translocase domain, is unable to initiate TCR. It strongly enhances the SOS response in the alkB(-)mfd(-) bacteria but not in the alkB(+)mfd(-) counterpart

TOPBP1 recruits TOP2A to ultra-fine anaphase bridges to aid in their resolution.

During mitosis, sister chromatids must be faithfully segregated to ensure that daughter cells receive one copy of each chromosome. However, following replication they often remain entangled. Topoisomerase IIα (TOP2A) has been proposed to resolve such entanglements, but the mechanisms governing TOP2A recruitment to these structures remain poorly understood. Here, we identify TOPBP1 as a novel interactor of TOP2A, and reveal that it is required for TOP2A recruitment to ultra-fine anaphase bridges (UFBs) in mitosis. The C-terminal region of TOPBP1 interacts with TOP2A, and TOPBP1 recruitment to UFBs requires its BRCT domain 5. Depletion of TOPBP1 leads to accumulation of UFBs, the majority of which arise from centromeric loci. Accordingly, expression of a TOPBP1 mutant that is defective in TOP2A binding phenocopies TOP2A depletion. These findings provide new mechanistic insights into how TOP2A promotes resolution of UFBs during mitosis, and highlights a pivotal role for TOPBP1 in this process.We thank Drs G. Stewart and F. Esashi for cell lines, Professor T.D. Halazonetis, Dr G.J. Gorbsky and Dr G. Stewart for plasmids and antibodies. We also thank Dr C. Lagerholm (Wolfson Imaging Centre, Oxford) and Dr D. Waithe (CBRG, Oxford) for their help with microscopy and image analysis, and the Mass Spectrometry Laboratory (IBB PAS) for their work on analyses of GFP–TOP2A immunoprecipitation experiments. We also thank Professor I. Hickson for helpful comments on the manuscript. This work was funded by a Worldwide Cancer Research International Fellowship (to W.N.), a WIMM/Medical Research Council Senior Non-Clinical Fellowship (to W.N.), a Polish Ministry of Science and Higher Education fellowship (to J.N.) and Polish National Science Center grant N N303 571539 (to J.N.).This is the final published version. It first appeared at http://www.nature.com/ncomms/2015/150312/ncomms7572/full/ncomms7572.html#abstract

TopBP1 interacts with BLM to maintain genome stability but is dispensable for preventing BLM degradation.

The Bloom syndrome helicase BLM and topoisomerase-IIβ-binding protein 1 (TopBP1) are key regulators of genome stability. It was recently proposed that BLM phosphorylation on Ser338 mediates its interaction with TopBP1, to protect BLM from ubiquitylation and degradation (Wang et al., 2013). Here, we show that the BLM-TopBP1 interaction does not involve Ser338 but instead requires BLM phosphorylation on Ser304. Furthermore, we establish that disrupting this interaction does not markedly affect BLM stability. However, BLM-TopBP1 binding is important for maintaining genome integrity, because in its absence cells display increased sister chromatid exchanges, replication origin firing and chromosomal aberrations. Therefore, the BLM-TopBP1 interaction maintains genome stability not by controlling BLM protein levels, but via another as-yet undetermined mechanism. Finally, we identify critical residues that mediate interactions between TopBP1 and MDC1, and between BLM and TOP3A/RMI1/RMI2. Taken together, our findings provide molecular insights into a key tumor suppressor and genome stability network.293FT cells, E1A antibody, and hr703 virus were gifts from Roger Grand, and DT40 cells and human LCLs were gifts from Julian Sale and Ian Hickson, respectively. We thank Nathan Ellis, Thanos Halazonetis, Frank Hänel, and Minoru Takata for plasmids; Grant Stewart and Yi Wang for antibodies; and Gabriel Balmus, Josep Forment, Abderrahmane Kaidi, Christine Schmidt, and Jon Travers for critical reading of the manuscript. This work was funded by a Worldwide Cancer Research International Fellowship and a WIMM/Medical Research Council Senior Non-Clinical Fellowship (MRCG0902418) to W.N., and by Polish Ministry of Science and Higher Education fellowship and Polish National Science Center grant number N303 571539 to J.N. The Jackson lab is funded by Cancer Research UK (CRUK) program grant C6/A11224, the European Research Council, and the European Community Seventh Framework Programme grant agreement number HEALTH-F2-2010-259893 (DDResponse). Core infrastructure funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, supplemented by CRUK.This is the final version of the article. It first appeared from Cell Press via http://dx.doi.org/10.1016/j.molcel.2015.02.01

Evaluation of the Escherichia coli HK82 and BS87 strains as tools for AlkB studies

Within a decade the family of AlkB dioxygenases has been extensively studied as a one-protein DNA/RNArepair system in Escherichia coli but also as a group of proteins of much wider functions in eukaryotes.Two strains, HK82 and BS87, are the most commonly used E. coli strains for the alkB gene mutations. Theaim of this study was to assess the usefulness of these alkB mutants in different aspects of research onAlkB dioxygenases that function not only in alkylated DNA repair but also in other metabolic processes incells. Using of HK82 and BS87 strains, we found the following differences among these alkB−derivatives:(i) HK82 has shown more than 10-fold higher MMS-induced mutagenesis in comparison to BS87; (ii)different specificity of Arg+revertants; (iii) increased induction of SOS and Ada responses in HK82; (iv)the genome of HK82, in comparison to AB1157 and BS87, contains additional mutations: nalA, sbcC, andnuoC. We hypothesize that in HK82 these mutations, together with the non-functional AlkB protein, mayresult in much higher contents of ssDNA, thus higher in comparison to BS87 MMS-induced mutagenesis.In the light of our findings, we strongly recommend using BS87 strain in AlkB research as HK82, bearingseveral additional mutations in its genome, is not an exact derivative of the AB1157 strain, and showsadditional features that may disturb proper interpretation of obtained results

Chloroacetaldehyde-induced mutagenesis in Escherichia coli: the role of AlkB protein in repair of 3,N4-ethenocytosine and 3,N4-α-hydroxyethanocytosine

Etheno () adducts are formed in reaction of DNA bases with various environmental carcinogens and endogenously created products of lipid peroxidation. Chloroacetaldehyde (CAA), a metabolite of carcinogen vinyl chloride, is routinely used to generate -adducts. We studied the role of AlkB, along with AlkA and Mug proteins, all engaged in repair of -adducts, in CAA-induced mutagenesis. The test system used involved pIF102 and pIF104 plasmids bearing the lactose operon of CC102 or CC104 origin (C.G. Cupples, J.H. Miller. Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 5345-5349) which allowed to monitor Lac+ revertants, the latter arose by GCAT or GCTA substitutions, respectively, as a result of modification of guanine and cytosine. The plasmids were CAA-damaged in vitro and replicated in E. coli of various genetic backgrounds. To modify the levels of AlkA and AlkB proteins, mutagenesis was studied in E. coli cells induced or not in adaptive response. Formation of C proceeds via a relatively stable intermediate, 3,N4--hydroxyethanocytosine (HEC), which allowed to compare repair of both adducts. The results indicate that all three genes, alkA, alkB and mug, are engaged in alleviation of CAA-induced mutagenesis. The frequency of mutation was higher in AlkA-, AlkB- and Mug-deficient strains in comparison to alkA+, alkB+, and mug+ controls. Considering the levels of CAA-induced Lac+ revertants in strains harboring the pIF plasmids and induced or not in adaptive response, we conclude that AlkB protein is engaged in the repair of C and HEC in vivo. Using the modified TTCTT 5-mers as substrates, we confirmed in vitro that AlkB protein repairs C and HEC although far less efficiently than the reference adduct 3-methylcytosine. The pH optimum for repair of HEC and εC is significantly different from that for 3-methylcytosine. We propose that the protonated form of adduct interact in active site of AlkB protein

Novel AlkB Dioxygenases—Alternative Models for In Silico and In Vivo Studies

Background: ALKBH proteins, the homologs of Escherichia coli AlkB dioxygenase, constitute a direct, single-protein repair system, protecting cellular DNA and RNA against the cytotoxic and mutagenic activity of alkylating agents, chemicals significantly contributing to tumor formation and used in cancer therapy. In silico analysis and in vivo studies have shown the existence of AlkB homologs in almost all organisms. Nine AlkB homologs (ALKBH1–8 and FTO) have been identified in humans. High ALKBH levels have been found to encourage tumor development, questioning the use of alkylating agents in chemotherapy. The aim of this work was to assign biological significance to multiple AlkB homologs by characterizing their activity in the repair of nucleic acids in prokaryotes and their subcellular localization in eukaryotes. Methodology and Findings: Bioinformatic analysis of protein sequence databases identified 1943 AlkB sequences with eight new AlkB subfamilies. Since Cyanobacteria and Arabidopsis thaliana contain multiple AlkB homologs, they were selected as model organisms for in vivo research. Using E. coli alkB2 mutant and plasmids expressing cyanobacterial AlkBs, we studied the repair of methyl methanesulfonate (MMS) and chloroacetaldehyde (CAA) induced lesions in ssDNA, ssRNA, and genomic DNA. On the basis of GFP fusions, we investigated the subcellular localization of ALKBHs in A. thaliana and established its mostly nucleo-cytoplasmic distribution. Some of the ALKBH proteins were found to change their localization upon MMS treatment. Conclusions: Our in vivo studies showed highly specific activity of cyanobacterial AlkB proteins towards lesions and nucleic acid type. Subcellular localization and translocation of ALKBHs in A. thaliana indicates a possible role for these proteins in the repair of alkyl lesions. We hypothesize that the multiplicity of ALKBHs is due to their involvement in the metabolism of nucleo-protein complexes; we find their repair by ALKBH proteins to be economical and effective alternative to degradation and de novo synthesis

EXD2 Protects Stressed Replication Forks and Is Required for Cell Viability in the Absence of BRCA1/2.

Accurate DNA replication is essential to preserve genomic integrity and prevent chromosomal instability-associated diseases including cancer. Key to this process is the cells' ability to stabilize and restart stalled replication forks. Here, we show that the EXD2 nuclease is essential to this process. EXD2 recruitment to stressed forks suppresses their degradation by restraining excessive fork regression. Accordingly, EXD2 deficiency leads to fork collapse, hypersensitivity to replication inhibitors, and genomic instability. Impeding fork regression by inactivation of SMARCAL1 or removal of RECQ1's inhibition in EXD2-/- cells restores efficient fork restart and genome stability. Moreover, purified EXD2 efficiently processes substrates mimicking regressed forks. Thus, this work identifies a mechanism underpinned by EXD2's nuclease activity, by which cells balance fork regression with fork restoration to maintain genome stability. Interestingly, from a clinical perspective, we discover that EXD2's depletion is synthetic lethal with mutations in BRCA1/2, implying a non-redundant role in replication fork protection.Work in W.N.’s laboratory is funded by ICR Intramural Grant and Cancer Research UK Programme (A24881). R.A.S. and L.S. were supported by WIMM Senior Non-Clinical Fellowship awarded to W.N. M.M.S. and M.A.B. were supported by the Intramural Research Program of the NIH, National Institute on Aging, United States (Z01-AG000746-08). Work in S.G.’s laboratory is supported by BRFAA Intramural Funds. V.C. was supported by John S. Latsis Public Benefit Foundation and Alexander S. Onassis Public Benefit Foundation. Work in the P.P.’s laboratory is supported by grants from the Agence Nationale pour la Recherche (ANR), the Ligue Contre le Cancer (équipe labellisée), SIRIC Montpellier Cancer (INCa Inserm DGOS 12553), and the MSDAvenir fund

Bacterial DNA repair genes and their eukaryotic homologues: 3. AlkB dioxygenase and Ada methyltransferase in the direct repair of alkylated DNA

Environmental and endogenous alkylating agents generate cytotoxic and mutagenic lesions in DNA. Exposure of prokaryotic cells to sublethal doses of DNA alkylating agents induces so called adaptive response (Ada response) involving the expression of a set of genes which allows the cells to tolerate the toxic and mutagenic action of such agents. The Ada response includes the expression of four genes: ada, alkA, alkB, and aidB. The product of ada gene, Ada protein, is an activator of transcription of all four genes. DNA bases damaged by alkylation are removed by distinct strategies. The most toxic lesion 3meA is removed by specific DNA glycosylase initiating base excising repair. The toxic and mutagenic O6meG is repaired directly by methyltransferases. 1meA and 3meC are corrected by AlkB DNA dioxygenase. The mechanisms of action of E. coli AlkB dioxygenase and its human homologs ABH2 and ABH3 are described in more details