DOCSLIB.ORG

Cleavage Mechanism of Human Mus81–Eme1 Acting on Holliday-Junction Structures

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Cleavage Mechanism of Human Mus81–Eme1 Acting on Holliday-Junction Structures Cleavage mechanism of human Mus81–Eme1 acting on Holliday-junction structures Ewan R. Taylor* and Clare H. McGowan*†‡ Departments of *Molecular Biology and †Cell Biology, The Scripps Research Institute, La Jolla, CA 92037 Edited by Stephen J. Elledge, Harvard Medical School, Boston, MA, and approved January 10, 2008 (received for review October 29, 2007) Recombination-mediated repair plays a central role in maintaining Results genomic integrity during DNA replication. The human Mus81–Eme1 Recombinant Human Mus81–Eme1. Truncation analysis of both endonuclease is involved in recombination repair, but the exact Mus81 and Eme1 was used to define the minimal domains structures it acts on in vivo are not known. Using kinetic and enzy- required for endonuclease function [see supplementary infor- matic analysis of highly purified recombinant enzyme, we find mation (SI) Fig. 5]. Versions of each protein containing amino that Mus81–Eme1 catalyzes coordinate bilateral cleavage of acids 260–551 for Mus81 and amino acids 244–571 for Eme1 model Holliday-junction structures. Using a self-limiting, cruciform- were expressed in Escherichia coli. The recombinant truncated containing substrate, we demonstrate that bilateral cleavage occurs complex, which we named EcME, was well expressed, largely sequentially within the lifetime of the enzyme–substrate complex. soluble, and, as detailed below, active. The complex was purified Coordinate bilateral cleavage is promoted by the highly cooperative to apparent homogeneity through affinity chromatography, ion nature of the enzyme and results in symmetrical cleavage of a exchange, and gel filtration steps (Fig. 1a). cruciform structure, thus, Mus81–Eme1 can ensure coordinate, bilat- eral cleavage of Holliday junction-like structures. Endonuclease Properties of a Minimal Mus81–Eme1 Complex. The activity of the recombinant enzyme was initially tested on three nuclease ͉ recombination repair model substrates, X12, nX12, and 3Ј flap, and each junction was radiolabeled on the 5Ј end of one oligonucleotide (Fig. 1b). The oligo nucleotide sequences are identical to those used previously (4, BIOCHEMISTRY he maintenance of genomic integrity requires multiple coordi- 16, 23). The X12 substrate has four 25-bp duplex DNA arms, with nated repair processes during DNA replication. Fork-stalling, T a 12-bp homologous core that allows branch migration of the recombination repair, and replication restart create a variety of junction. The nX12 substrate is identical to X12, except that it branched structures that are substrates for endonucleases. The contains a nick at the cross-over point. The 3Ј flap substrate Mus81–Eme1 endonuclease was first identified in budding yeast as contains 50 bp of duplex DNA, a central nick, and 25 bases of a mutant that causes sensitivity to replication-associated genotoxic single-stranded DNA. EcME is able to convert all three substrates stress (1). Fission yeast strains null for either Mus81 or Eme1 are to linear duplex product (Fig. 1b). The kinetic parameters of the exquisitely sensitive to replication stress and are inviable during EcME complex on these substrates were calculated by using meiosis (2, 3). Based on enzyme activity, damage sensitivity, and the nonlinear regression analysis of the reaction velocities obtained rescue of meiotic segregation defects by the prokaryotic resolvase, from experiments in which substrate concentration was varied (SI Ϫ1 RusA, a role in resolving Holliday junctions was proposed for Table 1 and SI Fig. 6). EcME has the highest kcat,0.18s , when Mus81–Eme1 in fission yeast (3). This function is supported by acting on an nX12 substrate. The enzyme functioned catalytically more recent data showing that the accumulation of X structures in in this reaction and converted 95 moles of substrate per mole of Mus81 delete cells in response to replication pausing and at sites of enzymein10min(SI Table 2). Consistent with previous observa- meiotic recombination (4, 5). tions using full-length Mus81–Eme1 (4, 24), EcME processed an Ϫ1 Mus81-null mice are viable, fertile, and have no obvious devel- intact X12 junction less efficiently, with a kcat of 0.0047 s . opmental defects (6, 7). Both mouse and human Mus81- and Nevertheless, at the highest concentration tested, EcME converted Eme1-null cell lines are exquisitely sensitive to interstrand crosslink Ͼ95% of the intact X12 substrate into linear duplex product (Fig. agents including mitomycin C (6–9). Mus81–Eme1 is recruited to 1b). The reduced catalytic efficiency of EcME on an intact X12 was sites of UV irradiation specifically during DNA replication (10). To driven both by a higher Km and a lower catalytic rate. The date, no meiotic defects have been reported in null Mus81 mice or observation that Mus81–Eme1 favors cleavage of nX12 over an X12 Drosophila, however, a role in generating interference-independent suggests that the initial cut on an X12 substrate is rate-limiting, and Ϸ crossovers has been reported for budding yeast and Arabidopsis (6, that it is followed by an 35-fold faster second cut. A nick- 7, 11–13). Also, Mus81 deficiency is lethal when combined with the counternick mechanism, in which a rate-limiting initial cut is disruption of the BLM helicase homologues in budding yeast, followed by a kinetically favored second-strand cleavage reaction, fission yeast, Drosophila, and Arabidopsis, suggesting a conserved has been used to explain the mechanism of action of a number of junction-resolving enzymes (4, 25–28). The kinetic parameters of role for Mus81–Eme1 in recombination repair and possibly Holli- Ј day-junction processing (2, 11, 14, 15). Data from several eukaryotic EcME on a 3 flap suggests that the complex binds this substrate organisms have shown that Mus81–Eme1 has activity on a number relatively poorly, but, once bound, the enzyme has a relatively robust rate of catalysis, with a k of 0.12 sϪ1. of branched DNA structures: Potential in vivo substrates are cat speculated to include forks, flaps, D-loops, and Holliday junctions (3, 4, 16–22). Author contributions: E.R.T. and C.H.M. designed research; E.R.T. performed research; In this study, we use highly purified recombinant Mus81–Eme1 E.R.T. and C.H.M. analyzed data; and E.R.T. and C.H.M. wrote the paper. to test the enzymatic properties and investigate the mechanism of The authors declare no conflict of interest. cleavage of model Holliday junctions. We define the catalytic core This article is a PNAS Direct Submission. of the enzyme complex. By using a plasmid-based substrate, we ‡To whom correspondence should be addressed. E-mail: [email protected]. demonstrate that Mus81–Eme1 uses a highly cooperative, coordi- This article contains supporting information online at www.pnas.org/cgi/content/full/ nated mechanism that ensures bilateral, symmetrical cleavage of 0710291105/DC1. Holliday-junction structures. © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710291105 PNAS ͉ March 11, 2008 ͉ vol. 105 ͉ no. 10 ͉ 3757–3762 Downloaded by guest on September 28, 2021 rations is explained by structural predications suggesting that these amino acids do not directly coordinate the catalytic cation and thus play a supporting rather than essential role in catalysis (29). The analogous mutation, D599A, in PfHef results in a similar dimin- ished, but not absent, nuclease activity (29). Substrate Specificity of Endogenous Mus81–Eme1 Endonuclease Ac- tivity. To test whether the truncations of Mus81 and Eme1 affect substrate preference, we compared the activity of EcME to full- length Mus81–Eme1 purified from baculovirus infected insect cells by anti-FLAG immunoprecipitation (Sf Mus81–Eme1). EcME has the same relative substrate preference as the recombinant full- length version of the protein, nX12 Ͼ 3Ј flap ϾϾ X12 (Fig. 1c), confirming that the truncations do not profoundly alter the sub- strate preference of the recombinant EcME enzyme. The activity profile of endogenous Mus81–Eme1 from a human cell line (HeLa) was also compared with that of recombinant full-length Mus81– Eme1 from insect cells (Fig. 1c). As with both recombinant versions of the complex, endogenous Mus81–Eme1 converted all three substrates into linear duplex (Fig. 1d). Notably, endogenous Mus81–Eme1 complex has a higher X12/nX12 activity ratio when compared with the recombinant protein. By comparing the amount of product generated at subsaturating levels of Mus81–Eme1 (i.e., 10% of immune precipitate), we find that the endogenous enzyme is only 3-fold less efficient on X12 than nX12 (Fig. 1d), whereas the recombinant insect cell-expressed enzyme is 10-fold less efficient (Fig. 1c). The endogenous enzyme had the same relative rate of Fig. 1. Endonuclease activities of recombinant human Mus81–Eme1. (a) activity when immune-precipitated with either monoclonal mouse Recombinant human Mus81–Eme1 purified from E. coli (EcME). Purified prep- arations were separated by SDS/PAGE and visualized by Coomassie staining. or polyclonal rabbit anti-Mus81 antibodies (data not shown). (b) Endonuclease activity of EcME on multiple substrates. The X12, nX12, and Gaskell et al. (31) recently concluded that full-length, recombi- 3Ј flap substrates, [200 pM], with 0.02, 0.2, 2, or 20 nM EcME for 20 min. nant, fission yeast Mus81–Eme1 has the same intact X versus nicked Products were separated by native PAGE. Substrates and DNA products are as X activity ratio as the endogenous protein. However, this compar- indicated. Each substrate is illustrated above the gels, and the 5Ј 32P radiolabel ison was based on assays done in two different laboratories (4, 31). is indicated by a circle. (c) Endonuclease activity of Myc.Mus81–Eme1.FLAG In this study, we compared the activity of recombinant full-length purified from Sf9 cells (Sf Mus81–Eme1). The indicated substrates [200 pM] human Mus81–Eme1 to the endogenous complex under identical were incubated with purified SfMus81–Eme1 for 20 min, with either 10%, assay conditions.
Load more

Recommended publications
  • SMX Makes the Cut in Genome Stability
    www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 61), pp: 102765-102766 Editorial SMX makes the cut in genome stability Haley D.M. Wyatt and Stephen C. West The faithful duplication and preservation of our SLX4 scaffold co-ordinate three different nucleases for genetic material is essential for cell survival; however, DNA cleavage? SMX was found to be a promiscuous DNA is susceptible to damage by extracellular and endonuclease that cleaves a broad range of DNA secondary intracellular agents (e.g. ultraviolet radiation, reactive structures in vitro3. Remarkably, SLX4 co-ordinates the oxygen species). DNA double-strand breaks (DSBs) SLX1 and MUS81-EME1 nucleases during HJ resolution are thought to represent the most dangerous type of [3, 4] (Figure 1). The involvement of two active sites from lesion, as the failure to repair a DSB can lead to loss of different heterodimeric enzymes leads to a non-canonical genetic information, chromosomal rearrangements, and mechanism of HJ resolution. It was also shown that SLX4 cell death. Fortunately, cells contain sophisticated DNA activates MUS81-EME1 to cleave structures that resemble repair pathways to counteract the deleterious effects of stalled replication forks [3] (Figure 1). Activation involves genotoxic agents. Mutations in DNA repair genes are relaxation of MUS81-EME1’s substrate specificity, which linked to various diseases, including neurological defects, is regulated by a helix-hairpin-helix (HhH) domain in immunodeficiency, and cancer. the MUS81 N-terminus (MUS81 N-HhH). Intriguingly, Gross chromosomal rearrangements, including MUS81 N-HhH also mediates the interaction with deletions, duplications, and translocations, are a hallmark SLX4 via a C-terminal SAP domain (SLX4 SAP) [5].
    [Show full text]
  • The Choice in Meiosis – Defining the Factors That Influence Crossover Or Non-Crossover Formation
    Commentary 501 The choice in meiosis – defining the factors that influence crossover or non-crossover formation Jillian L. Youds and Simon J. Boulton* DNA Damage Response Laboratory, Cancer Research UK, London Research Institute, Clare Hall, Blanche Lane, South Mimms EN6 3LD, UK *Author for correspondence ([email protected]) Journal of Cell Science 124, 501-513 © 2011. Published by The Company of Biologists Ltd doi:10.1242/jcs.074427 Summary Meiotic crossovers are essential for ensuring correct chromosome segregation as well as for creating new combinations of alleles for natural selection to take place. During meiosis, excess meiotic double-strand breaks (DSBs) are generated; a subset of these breaks are repaired to form crossovers, whereas the remainder are repaired as non-crossovers. What determines where meiotic DSBs are created and whether a crossover or non-crossover will be formed at any particular DSB remains largely unclear. Nevertheless, several recent papers have revealed important insights into the factors that control the decision between crossover and non-crossover formation in meiosis, including DNA elements that determine the positioning of meiotic DSBs, and the generation and processing of recombination intermediates. In this review, we focus on the factors that influence DSB positioning, the proteins required for the formation of recombination intermediates and how the processing of these structures generates either a crossover or non-crossover in various organisms. A discussion of crossover interference, assurance and homeostasis, which influence crossing over on a chromosome-wide and genome-wide scale – in addition to current models for the generation of interference – is also included. This Commentary aims to highlight recent advances in our understanding of the factors that promote or prevent meiotic crossing over.
    [Show full text]
  • Regulation of Mus81-Eme1 Structure-Specific Endonuclease by Eme1 SUMO-Binding and Rad3atr Kinase Is Essential in the Absence of Rqh1blm Helicase
    bioRxiv preprint doi: https://doi.org/10.1101/2021.07.29.454171; this version posted July 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Regulation of Mus81-Eme1 structure-specific endonuclease by Eme1 SUMO-binding and Rad3ATR kinase is essential in the absence of Rqh1BLM helicase Giaccherini C.1,#, Scaglione S. 1, Coulon S. 1, Dehé P.M.1,#,* and Gaillard P.H.L. 1* 1 Centre de Recherche en Cancérologie de Marseille, CRCM, Inserm, CNRS, Aix- Marseille Université, Institut Paoli-Calmettes, Marseille, France. #The authors contributed equally *correspondence [email protected] [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.07.29.454171; this version posted July 29, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract The Mus81-Eme1 structure-specific endonuclease is crucial for the processing of DNA recombination and late replication intermediates. In fission yeast, stimulation of Mus81-Eme1 in response to DNA damage at the G2/M transition relies on Cdc2CDK1 and DNA damage checkpoint-dependent phosphorylation of Eme1 and is critical for chromosome stability in absence of the Rqh1BLM helicase. Here we identify Rad3ATR checkpoint kinase consensus phosphorylation sites and two SUMO interacting motifs (SIM) within a short N-terminal domain of Eme1 that is required for cell survival in absence of Rqh1BLM.
    [Show full text]
  • The MUS81 Endonuclease Is Essential for Telomerase Negative Cell Proliferation Sicong Zeng Washington University School of Medicine in St
    Washington University School of Medicine Digital Commons@Becker Open Access Publications 2009 The MUS81 endonuclease is essential for telomerase negative cell proliferation Sicong Zeng Washington University School of Medicine in St. Louis Qin Yang Washington University School of Medicine in St. Louis Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs Recommended Citation Zeng, Sicong and Yang, Qin, ,"The UM S81 endonuclease is essential for telomerase negative cell proliferation." Cell Cycle.8,15. 2157-2160. (2009). https://digitalcommons.wustl.edu/open_access_pubs/3006 This Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in Open Access Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact [email protected]. [Cell Cycle 8:15, 2157-2160; 1 August 2009]; ©2009 Landes Bioscience Extra View The MUS81 endonuclease is essential for telomerase negative cell proliferation Sicong Zeng† and Qin Yang* Department of Radiation Oncology; Washington University School of Medicine; St. Louis, MO USA †Present address: Institute of Reproduction and Stem Cell Engineering; Central South University; Changsha, Hunan China Key words: ALT, APBs, MUS81, telomere recombination A substantial number of human tumors (~10%) are telomerase breaks and maintains telomere length and stability. Progressive negative, and cells in such tumors have been proposed to main- telomere shortening in normal cells during DNA replication leads tain telomere length by the alternative lengthening of telomeres eventually to a permanent halt of cell division referred to as replica- (ALT) pathway. Although details of the molecular mechanism tive senescence. This end replication problem of human telomeres of ALT are largely unknown, previous studies have shown that has received particular attention due to its implications in ageing telomere homologous recombination (HR) is implicated in the and cancer.
    [Show full text]
  • Structure of the Germline Genome of Tetrahymena Thermophila
    1 Structure of the germline genome of Tetrahymena thermophila 2 and relationship to the massively rearranged somatic genome 3 4 Eileen P. Hamiltona#, Aurélie Kapustab#, Piroska E. Huvosc, Shelby L. Bidwelld, Nikhat Zafard, 5 Haibao Tangd, Michalis Hadjithomasd, Vivek Krishnakumard, Jonathan Badgerd, Elisabet V. 6 Calerd, Carsten Russe, Qiandong Zenge, Lin Fane, Joshua Z. Levine, Terrance Sheae, Sarah K. 7 Younge, Ryan Hegartye, Riza Dazae, Sharvari Gujjae, Jennifer R. Wortmane, Bruce Birrene, 8 Chad Nusbaume, Jainy Thomasb, Clayton M. Careyb, Ellen J. Prithamb, Cédric Feschotteb, 9 Tomoko Notof, Kazufumi Mochizukif, Romeo Papazyang, Sean D. Tavernag, Paul H. Dearh, 10 Donna M. Cassidy-Hanleyi, Jie Xiongj, Wei Miaoj, Eduardo Oriasa, Robert S. Coyned* 11 12 a: Department of Molecular, Cellular, and Developmental Biology, University of California, 13 Santa Barbara, CA, USA 14 b: Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT, 15 USA 16 c: Department of Medical Biochemistry, Southern Illinois University, Carbondale, IL, USA. 17 d: J. Craig Venter Institute, Rockville, MD, USA 18 e: Eli and Edythe L. Broad Institute of Harvard and MIT, Cambridge, MA, USA 19 f: Institute of Molecular Biotechnology, Vienna, Austria; current address: Institute of 20 Human Genetics - CNRS, Montpellier, France 21 g: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University 22 School of Medicine, Baltimore, MD, USA 1 23 h: MRC Laboratory of Molecular Biology, Cambridge, UK 24 i: Dept. of Microbiology and Immunology, Cornell University Veterinary Medical Center, 25 Ithaca, NY, USA 26 j: Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, People's Republic of 27 China 28 *: corresponding author.
    [Show full text]
  • Roles of SLX1–SLX4, MUS81–EME1, and GEN1 in Avoiding Genome Instability and Mitotic Catastrophe
    Downloaded from genesdev.cshlp.org on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press Roles of SLX1–SLX4, MUS81–EME1, and GEN1 in avoiding genome instability and mitotic catastrophe Shriparna Sarbajna,1 Derek Davies,2 and Stephen C. West1,3 1Clare Hall Laboratories, Cancer Research UK, London Research Institute, Herts EN6 3LD, United Kingdom; 2London Research Institute, London WC2A 3PX, United Kingdom The resolution of recombination intermediates containing Holliday junctions (HJs) is critical for genome maintenance and proper chromosome segregation. Three pathways for HJ processing exist in human cells and involve the following enzymes/complexes: BLM–TopoIIIa–RMI1–RMI2 (BTR complex), SLX1–SLX4–MUS81– EME1 (SLX–MUS complex), and GEN1. Cycling cells preferentially use the BTR complex for the removal of double HJs in S phase, with SLX–MUS and GEN1 acting at temporally distinct phases of the cell cycle. Cells lacking SLX–MUS and GEN1 exhibit chromosome missegregation, micronucleus formation, and elevated levels of 53BP1-positive G1 nuclear bodies, suggesting that defects in chromosome segregation lead to the transmission of extensive DNA damage to daughter cells. In addition, however, we found that the effects of SLX4, MUS81, and GEN1 depletion extend beyond mitosis, since genome instability is observed throughout all phases of the cell cycle. This is exemplified in the form of impaired replication fork movement and S-phase progression, endogenous checkpoint activation, chromosome segmentation, and multinucleation. In contrast to SLX4, SLX1, the nuclease subunit of the SLX1–SLX4 structure-selective nuclease, plays no role in the replication-related phenotypes associated with SLX4/MUS81 and GEN1 depletion.
    [Show full text]
  • Msh4 and Msh5 Function in SC-Independent Chiasma Formation During the Streamlined Meiosis of Tetrahymena
    HIGHLIGHTED ARTICLE INVESTIGATION Msh4 and Msh5 Function in SC-Independent Chiasma Formation During the Streamlined Meiosis of Tetrahymena Anura Shodhan,* Agnieszka Lukaszewicz,* Maria Novatchkova,†,‡ and Josef Loidl*,1 *Department of Chromosome Biology and Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, A-1030 Vienna, Austria, †Research Institute of Molecular Pathology, A-130 Vienna, Austria, and ‡Institute of Molecular Biotechnology of the Austrian Academy of Sciences, A-1030 Vienna, Austria ORCID ID: 0000-0002-3675-2146 (A.S.) ABSTRACT ZMM proteins have been defined in budding yeast as factors that are collectively involved in the formation of interfering crossovers (COs) and synaptonemal complexes (SCs), and they are a hallmark of the predominant meiotic recombination pathway of most organisms. In addition to this so-called class I CO pathway, a minority of crossovers are formed by a class II pathway, which involves the Mus81-Mms4 endonuclease complex. This is the only CO pathway in the SC-less meiosis of the fission yeast. ZMM proteins (including SC components) were always found to be co-occurring and hence have been regarded as functionally linked. Like the fission yeast, the protist Tetrahymena thermophila does not possess a SC, and its COs are dependent on Mus81-Mms4. Here we show that the ZMM proteins Msh4 and Msh5 are required for normal chiasma formation, and we propose that they have a pro-CO function outside a canonical class I pathway in Tetrahymena. Thus, the two-pathway model is not tenable as a general rule. EIOSIS is a specialized cell division by which the dip- half in budding yeast (Mancera et al.
    [Show full text]
  • ATR Protects the Genome Against R Loops Through a MUS81-Triggered Feedback Loop
    ATR Protects the Genome against R loops through a MUS81-Triggered Feedback Loop The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Matos, Dominick Amaral. 2020. ATR Protects the Genome against R loops through a MUS81-Triggered Feedback Loop. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link https://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37365155 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA ATR Protects the Genome against R loops through a MUS81-Triggered Feedback Loop A dissertation presented by Dominick A Matos to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biological and Biomedical Sciences Harvard University Cambridge, Massachusetts December 2019 @ 2019 Dominick A Matos All rights reserved. Dissertation Advisor: Lee Zou Dominick A Matos ATR Protects the Genome against R loops through a MUS81-Triggered Feedback Loop Abstract R loops are three-stranded transcription intermediates consisting of RNA:DNA hybrids and displaced single-stranded DNA. Although R loop formation is important for a number of cellular processes, they can cause genomic instability if dysregulated. The R loop-induced genomic instability is largely dependent on DNA replication, suggesting that it may arise from the collisions between R loops and DNA replication forks. How cells respond to the collisions between R loops and replication fork is poorly understood.
    [Show full text]
  • Two Distinct MUS81-EME1 Complexes from Arabidopsis Thaliana Process
    Two Distinct MUS81-EME1 Complexes from Arabidopsis Process Holliday Junctions1[W] Verena Geuting2, Daniela Kobbe, Frank Hartung, Jasmin Du¨ rr3, Manfred Focke, and Holger Puchta* Botanik II, Universita¨t Karlsruhe, 76128 Karlsruhe, Germany The MUS81 endonuclease complex has been shown to play an important role in the repair of stalled or blocked replication forks and in the processing of meiotic recombination intermediates from yeast to humans. This endonuclease is composed of two subunits, MUS81 and EME1. Surprisingly, unlike other organisms, Arabidopsis (Arabidopsis thaliana) has two EME1 homologs encoded in its genome. AtEME1A and AtEME1B show 63% identity on the protein level. We were able to demonstrate that, after expression in Escherichia coli, each EME1 protein can assemble with the unique AtMUS81 to form a functional endonuclease. Both complexes, AtMUS81-AtEME1A and AtMUS81-AtEME1B, are not only able to cleave 3#-flap structures and nicked Holliday junctions (HJs) but also, with reduced efficiency, intact HJs. While the complexes have the same cleavage patterns with both nicked DNA substrates, slight differences in the processing of intact HJs can be detected. Our results are in line with an involvement of both MUS81-EME1 endonuclease complexes in DNA recombination and repair processes in Arabidopsis. DNA is constantly damaged by extrinsic and intrin- nucleases and forms a functional endonuclease com- sic factors, such as UV and ionizing irradiation, chem- plex with its interaction partner EME1 (also referred to icals, and cellular processes. The accumulation of as MMS4 in Saccharomyces cerevisiae; Boddy et al., 2000; DNA damage can impair essential processes, includ- Interthal and Heyer, 2000; Chen et al., 2001).
    [Show full text]
  • Bi-Allelic MCM10 Variants Associated with Immune Dysfunction and Cardiomyopathy Cause Telomere Shortening
    ARTICLE https://doi.org/10.1038/s41467-021-21878-x OPEN Bi-allelic MCM10 variants associated with immune dysfunction and cardiomyopathy cause telomere shortening Ryan M. Baxley 1, Wendy Leung1,11, Megan M. Schmit 1,11, Jacob Peter Matson 2,11, Lulu Yin1, Marissa K. Oram 1, Liangjun Wang1, John Taylor3, Jack Hedberg1, Colette B. Rogers1, Adam J. Harvey1, Debashree Basu1, Jenny C. Taylor4,5, Alistair T. Pagnamenta 4,5, Helene Dreau6, Jude Craft7, Elizabeth Ormondroyd 8, Hugh Watkins 8, Eric A. Hendrickson 1, Emily M. Mace 9, Jordan S. Orange9, ✉ Hideki Aihara 1, Grant S. Stewart 10, Edward Blair7, Jeanette Gowen Cook 2 & Anja-Katrin Bielinsky 1 1234567890():,; Minichromosome maintenance protein 10 (MCM10) is essential for eukaryotic DNA repli- cation. Here, we describe compound heterozygous MCM10 variants in patients with dis- tinctive, but overlapping, clinical phenotypes: natural killer (NK) cell deficiency (NKD) and restrictive cardiomyopathy (RCM) with hypoplasia of the spleen and thymus. To understand the mechanism of MCM10-associated disease, we modeled these variants in human cell lines. MCM10 deficiency causes chronic replication stress that reduces cell viability due to increased genomic instability and telomere erosion. Our data suggest that loss of MCM10 function constrains telomerase activity by accumulating abnormal replication fork structures enriched with single-stranded DNA. Terminally-arrested replication forks in MCM10- deficient cells require endonucleolytic processing by MUS81, as MCM10:MUS81 double mutants display decreased viability and accelerated telomere shortening. We propose that these bi-allelic variants in MCM10 predispose specific cardiac and immune cell lineages to prematurely arrest during differentiation, causing the clinical phenotypes observed in both NKD and RCM patients.
    [Show full text]
  • Supporting Information
    Supporting Information Cnossen et al. 10.1073/pnas.1309438111 SI Text gs.washington.edu/EVS/ (in 6,500 individuals variants were Investigations in Polycystic Liver Disease-1 Family. Ultrasonography. assessed in the project) Ultrasound images of liver and kidneys were acquired using Database of Single Nucleotide Polymorphisms (dbSNP): www. a 3.6-MHz general purpose clinical echo system (Acuson ×150; ncbi.nlm.nih.gov/projects/SNP/; Bethesda (MD): National Cen- Siemens AG) equipped with a curved linear array transducer in ter for Biotechnology Information, National Library of Medi- all 39 members of proband III/18. Presence or absence of hepatic cine; NCBI dbSNP Build 137; 26th June 2012 available and/or renal cysts was investigated and noted carefully by at least Mouse Genome Informatics; v.MGI 5.12 (last database up- two clinicians (W.R.C., M.C., and J.P.H.D.). These ultrasound date 03–13-2013): www.informatics.jax.org/searches/allele_ examinations were conducted in all 39 members within 2 wk. report.cgi?_Marker_key=37359;MGD During the project, five members (II/13; III/13; III/14; III/21; and Human gene mutation database (HGMD Professional) (www. III/26) were reevaluated by abdominal ultrasonography (Table biobase-international.com/hgmd) from BIOBASE Corpora- S4). Member III/22 could not undergo a second evaluation due tion; Professional 2012.4, 14 December 2012 accessed to chronic health issues. A complete clinical analysis of the liver MRS database; v.6 (latest version): http://mrs.cmbi.ru.nl/m6/ and kidney phenotype in these individuals by CT or MRI scan- entry?db=sprot&id=lrp5_human&rq=lrp5_human ning was not ethically approved for this project.
    [Show full text]
  • Structure-Specific Endonucleases and the Resolution of Chromosome Underreplication
    G C A T T A C G G C A T genes Review Structure-Specific Endonucleases and the Resolution of Chromosome Underreplication Benoît Falquet 1,2 and Ulrich Rass 3,* 1 Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland; [email protected] 2 Faculty of Natural Sciences, University of Basel, Petersplatz 10, CH-4003 Basel, Switzerland 3 Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9RQ, UK * Correspondence: [email protected] Received: 22 February 2019; Accepted: 13 March 2019; Published: 19 March 2019 Abstract: Complete genome duplication in every cell cycle is fundamental for genome stability and cell survival. However, chromosome replication is frequently challenged by obstacles that impede DNA replication fork (RF) progression, which subsequently causes replication stress (RS). Cells have evolved pathways of RF protection and restart that mitigate the consequences of RS and promote the completion of DNA synthesis prior to mitotic chromosome segregation. If there is entry into mitosis with underreplicated chromosomes, this results in sister-chromatid entanglements, chromosome breakage and rearrangements and aneuploidy in daughter cells. Here, we focus on the resolution of persistent replication intermediates by the structure-specific endonucleases (SSEs) MUS81, SLX1-SLX4 and GEN1. Their actions and a recently discovered pathway of mitotic DNA repair synthesis have emerged as important facilitators of replication completion and sister chromatid detachment in mitosis. As RS is induced by oncogene activation and is a common feature of cancer cells, any advances in our understanding of the molecular mechanisms related to chromosome underreplication have important biomedical implications.
    [Show full text]