DOCSLIB.ORG

Holliday Junction-Containing DNA Structures Persist in Cells Lacking Sgs1 Or Top3 Following Exposure to DNA Damage

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Holliday Junction-Containing DNA Structures Persist in Cells Lacking Sgs1 Or Top3 Following Exposure to DNA Damage Holliday junction-containing DNA structures persist in cells lacking Sgs1 or Top3 following exposure to DNA damage Hocine W. Mankouria,b,1, Thomas M. Ashtona,1, and Ian D. Hicksona,b,2 aWeatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom; and bNordea Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen, 2200 Copenhagen N, Denmark Edited* by Stephen C. Kowalczykowski, University of California, Davis, CA, and approved February 14, 2011 (received for review September 22, 2010) The Sgs1–Rmi1–Top3 “dissolvasome” is required for the mainte- HRR is a conserved cellular process that allows cells to copy nance of genome stability and has been implicated in the process- genetic information from a homologous sequence, and is re- ing of various types of DNA structures arising during DNA re- quired for the efficient repair of DNA breaks and ssDNA gaps plication. Previous investigations have revealed that unprocessed that can arise during S-phase due to DNA damage, discon- (X-shaped) homologous recombination repair (HRR) intermediates tinuities in DNA replication, or the impediment of replication persist when S-phase is perturbed by using methyl methanesulfo- fork (RF) progression. Evidence that BLM functions in HRR is nate (MMS) in Saccharomyces cerevisiae cells with impaired Sgs1 suggested by the fact that BS cells demonstrate elevated levels of or Top3. However, the precise nature of these persistent DNA struc- mitotic recombination, sister chromatid exchanges, and genome instability (1). Furthermore, BLM (either alone or in conjunction tures remains poorly characterized. Here, we report that ectopic α expression of either of two heterologous and structurally unrelated with hTOPOIII , hRMI1, and hRMI2) can resolve different types Escherichia coli of HRR intermediates in vitro, such as D-loops and single or Holliday junction (HJ) resolvases, RusA or human – GEN11-527, promotes the removal of these X-structures in vivo. double Holliday junctions (HJs; refs. 6, 7, and 16 21). Mutation of SGS1 or the Schizosaccharomyces pombe ortholog of BLM/SGS1, Moreover, other types of DNA replication intermediates, including + called rqh1 , also causes genome instability, hyperrecombination, GENETICS stalled replication forks and non-HRR-dependent X-structures, are and sensitivity to DNA-damaging agents (8, 22–25), and Sgs1, like refractory to RusA or GEN11-527, demonstrating specificity of these BLM, is able to unwind HJs in vitro (26). Furthermore, un- HJ resolvases for MMS-induced X-structures in vivo. These data processed HRR intermediates (X-structures) have been directly suggest that the X-structures persisting in cells with impaired observed in methyl methanesulfonate (MMS)-treated sgs1, top3, Sgs1 or Top3 contain HJs. Furthermore, we demonstrate that Sgs1 and rmi1 mutants using 2D gel electrophoresis (27–30). Because directly promotes X-structure removal, because the persistent many of the deleterious phenotypes of sgs1 or rqh1 mutants can be fi structures arising in Sgs1-de cient strains are eliminated when suppressed by the mutation of genes involved in the early steps of Sgs1 is reactivated in vivo. We propose that HJ resolvases and HRR (e.g., RAD51 in S. cerevisiae and rhp51+ in S. pombe; refs. Sgs1–Top3–Rmi1 comprise two independent processes to deal with 31–35), it is likely that these phenotypes are, at least in part, due to HJ-containing DNA intermediates arising during HRR in S-phase. unregulated or incomplete HRR. Although X-shaped HRR structures have been detected by DNA repair | RecQ | helicase | topoisomerase using 2D DNA gel electrophoresis in S. cerevisiae cells with im- paired Sgs1, Top3, or Rmi1 (27–30), it is presently unknown he RecQ family of DNA helicases is required for the main- whether different types of DNA structures arise in cells deficient Ttenance of genome stability in all organisms. Mutations in at in these different proteins. Because abolition of Sgs1 activity (or least three (of five) human RecQ helicases is associated with an just its helicase activity) can suppress the poor growth pheno- increased predisposition to the development of cancer and/or types of top3 or rmi1 mutants (8, 13, 14, 36), it has been proposed premature aging (1). Mutations in BLM cause Bloom’s syndrome that Sgs1 may create a DNA intermediate that is toxic in cells (BS), which is associated with increased cancer predisposition, lacking Top3 or Rmi1. One proposal is that the convergent whereas mutations in WRN or RECQ4 cause distinct disorders branch migration of double HJs (dHJs) by Sgs1 creates a hemi- (Werner’s syndrome and Rothmund–Thomson syndrome, re- catenane structure that can only be resolved by Top3 (in con- spectively) characterized by premature aging and some de- junction with Rmi1), in a process known as “dHJ dissolution” velopmental abnormalities. In Saccharomyces cerevisiae, there is (18, 20, 21). However, in vivo evidence for this process is lacking, only one RecQ helicase, called Sgs1. As a consequence, this because the 2D gel methodology cannot definitively distinguish organism has proved particularly useful for genetic analyses, as between different types of joint DNA molecules such as HJs and well as for characterization of the defects arising in cells lacking hemicatenanes. RecQ helicases (2). The X-structures arising in MMS-treated sgs1 mutants have “ ” Sgs1 is thought to be the ortholog of BLM, based on a number been proposed to be pseudo-HJs, consisting of a region of of observations. First, BLM is the only human RecQ helicase hemicatenated nascent DNA and concomitant single-stranded that shares the same structural domain architecture as Sgs1. regions of parental DNA (27). This conclusion was based on the Second, both BLM and Sgs1 associate with conserved interacting in vitro characterization of X-structures, including their ability to partners that are apparently unique for BLM in human cells. More specifically, BLM exists in a complex with a type IA topoisom- erase, hTOPOIIIα, and two oligonucleotide/oligosaccharide- Author contributions: H.W.M. and I.D.H. designed research; H.W.M. and T.M.A. per- binding (OB)-fold containing proteins, hRMI1 and hRMI2 (3– formed research; H.W.M. contributed new reagents/analytic tools; H.W.M., T.M.A., and 7); Sgs1 associates with the yeast orthologs of these proteins, I.D.H. and analyzed data; and H.W.M. wrote the paper. Top3 and Rmi1 (8–14). Together, these proteins likely act co- The authors declare no conflict of interest. operatively as a “dissolvasome” that processes multiple kinds of *This Direct Submission article had a prearranged editor. DNA structures arising during DNA replication, repair, recom- 1H.W.M. and T.M.A. contributed equally to this work. bination, and mitosis (15). 2To whom correspondence should be addressed. E-mail: [email protected]. One cellular process in which BLM and Sgs1 have both been This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. strongly implicated is homologous recombination repair (HRR). 1073/pnas.1014240108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1014240108 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 branch migrate (even in the presence of Mg2+, which generally medium containing MMS or HU at 30 °C for 1 h. Cultures were inhibits HJ migration), their apparent resistance to the RuvC HJ then incubated at 37 °C for a further 6 h to promote robust HJ resolvase, and their partial sensitivity to ssDNA nucleases (27). resolvase activity. Genomic DNA was extracted by using the MMS also causes increased interhomolog X-structures in sgs1 hexadecyltrimethylammonium bromide (CTAB) method to re- diploid cells (37), although the precise nature of these in- strain branch migration of joint (X-shaped) molecules (42). Af- terchromosomal joint DNA molecules remains to be determined. ter 7-h exposure to 0.033% MMS, all DNA replication inter- Interestingly, a recent study demonstrated that interchromosomal mediates had disappeared from ARS305 in WT cells expressing dHJs arise during double-strand break (DSB) repair in mitotic GFP, consistent with the completion of DNA replication at this diploid cells (38). Furthermore, the turnover of these structures region of the genome by this stage. In sgs1 mutants expressing was altered in sgs1 mutants, consistent with a possible role for Sgs1 GFP, bubbles and Y arcs had also disappeared, but, consistent in the prevention and/or direct processing of HJs in vivo (38). In- with previous findings (27, 28, 30), persistent X-structures at ter-sister chromatid X-structures were also identified as inter- ARS305 were observed (Fig. 1B). Previous analyses have iden- mediates of DSB repair in this study, although whether these tified these structures to be Rad51-dependent HRR inter- contained HJs or not could not be determined. Therefore, despite mediates (27). Interestingly, these MMS-induced X-structures several independent studies demonstrating the existence of un- were not detectable in sgs1 cells expressing RusA and GEN11-527 processed X-shaped DNA structures in sgs1 mutants under a (Fig. 1B). All strains examined exhibited similar mid-S-phase number of different experimental conditions, it remains unknown FACS profiles (Fig. S1), suggesting that the observed differences whether these comprise a common type of DNA structure or not. on 2D gels were not attributable to any differences in cell cycle Indeed, the precise nature and abundance of the X-structures progression or MMS-induced checkpoint arrest. We conclude could vary depending on the context (e.g., in DSB repair vs. the that expression of RusA and GEN11-527 leads to a marked re- repair of replication-associated lesions). Consistent with this pro- duction in the level of unprocessed HRR intermediates in MMS- posal, RecQ helicases can process a variety of different DNA treated sgs1 cells. structures in addition to HJs in vitro, and the BLM–hTOPOIIIα– hRMI1–hRMI2 and Sgs1–Top3–Rmi1 complexes probably act as Stalled RFs and Non-HRR-Dependent X-Structures Are Refractory to DNA structure-specific “dissolvasomes” in vivo (15).
Load more

Recommended publications
  • Structure and Function of the Human Recq DNA Helicases
    Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2005 Structure and function of the human RecQ DNA helicases Garcia, P L Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-34420 Dissertation Published Version Originally published at: Garcia, P L. Structure and function of the human RecQ DNA helicases. 2005, University of Zurich, Faculty of Science. Structure and Function of the Human RecQ DNA Helicases Dissertation zur Erlangung der naturwissenschaftlichen Doktorw¨urde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultat¨ der Universitat¨ Z ¨urich von Patrick L. Garcia aus Unterseen BE Promotionskomitee Prof. Dr. Josef Jiricny (Vorsitz) Prof. Dr. Ulrich H ¨ubscher Dr. Pavel Janscak (Leitung der Dissertation) Z ¨urich, 2005 For my parents ii Summary The RecQ DNA helicases are highly conserved from bacteria to man and are required for the maintenance of genomic stability. All unicellular organisms contain a single RecQ helicase, whereas the number of RecQ homologues in higher organisms can vary. Mu- tations in the genes encoding three of the five human members of the RecQ family give rise to autosomal recessive disorders called Bloom syndrome, Werner syndrome and Rothmund-Thomson syndrome. These diseases manifest commonly with genomic in- stability and a high predisposition to cancer. However, the genetic alterations vary as well as the types of tumours in these syndromes. Furthermore, distinct clinical features are observed, like short stature and immunodeficiency in Bloom syndrome patients or premature ageing in Werner Syndrome patients. Also, the biochemical features of the human RecQ-like DNA helicases are diverse, pointing to different roles in the mainte- nance of genomic stability.
    [Show full text]
  • Open Full Page
    CCR PEDIATRIC ONCOLOGY SERIES CCR Pediatric Oncology Series Recommendations for Childhood Cancer Screening and Surveillance in DNA Repair Disorders Michael F. Walsh1, Vivian Y. Chang2, Wendy K. Kohlmann3, Hamish S. Scott4, Christopher Cunniff5, Franck Bourdeaut6, Jan J. Molenaar7, Christopher C. Porter8, John T. Sandlund9, Sharon E. Plon10, Lisa L. Wang10, and Sharon A. Savage11 Abstract DNA repair syndromes are heterogeneous disorders caused by around the world to discuss and develop cancer surveillance pathogenic variants in genes encoding proteins key in DNA guidelines for children with cancer-prone disorders. Herein, replication and/or the cellular response to DNA damage. The we focus on the more common of the rare DNA repair dis- majority of these syndromes are inherited in an autosomal- orders: ataxia telangiectasia, Bloom syndrome, Fanconi ane- recessive manner, but autosomal-dominant and X-linked reces- mia, dyskeratosis congenita, Nijmegen breakage syndrome, sive disorders also exist. The clinical features of patients with DNA Rothmund–Thomson syndrome, and Xeroderma pigmento- repair syndromes are highly varied and dependent on the under- sum. Dedicated syndrome registries and a combination of lying genetic cause. Notably, all patients have elevated risks of basic science and clinical research have led to important in- syndrome-associated cancers, and many of these cancers present sights into the underlying biology of these disorders. Given the in childhood. Although it is clear that the risk of cancer is rarity of these disorders, it is recommended that centralized increased, there are limited data defining the true incidence of centers of excellence be involved directly or through consulta- cancer and almost no evidence-based approaches to cancer tion in caring for patients with heritable DNA repair syn- surveillance in patients with DNA repair disorders.
    [Show full text]
  • Helicase Mechanisms During Homologous Recombination in Saccharomyces Cerevisiae
    BB48CH11_Greene ARjats.cls April 18, 2019 12:24 Annual Review of Biophysics Helicase Mechanisms During Homologous Recombination in Saccharomyces cerevisiae J. Brooks Crickard and Eric C. Greene Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA; email: [email protected], [email protected] Annu. Rev. Biophys. 2019. 48:255–73 Keywords First published as a Review in Advance on homologous recombination, helicase, Srs2, Sgs1, Rad54 March 11, 2019 Access provided by 68.175.70.229 on 06/02/20. For personal use only. The Annual Review of Biophysics is online at Abstract Annu. Rev. Biophys. 2019.48:255-273. Downloaded from www.annualreviews.org biophys.annualreviews.org Helicases are enzymes that move, manage, and manipulate nucleic acids. https://doi.org/10.1146/annurev-biophys-052118- They can be subdivided into six super families and are required for all aspects 115418 of nucleic acid metabolism. In general, all helicases function by converting Copyright © 2019 by Annual Reviews. the chemical energy stored in the bond between the gamma and beta phos- All rights reserved phates of adenosine triphosphate into mechanical work, which results in the unidirectional movement of the helicase protein along one strand of a nu- cleic acid. The results of this translocation activity can range from separation of strands within duplex nucleic acids to the physical remodeling or removal of nucleoprotein complexes. In this review, we focus on describing key heli- cases from the model organism Saccharomyces cerevisiae that contribute to the regulation of homologous recombination, which is an essential DNA repair pathway for fxing damaged chromosomes.
    [Show full text]
  • The Role of SLX4 and Its Associated Nucleases in DNA Interstrand Crosslink Repair Wouter S
    Nucleic Acids Research, 2018 1 doi: 10.1093/nar/gky1276 The role of SLX4 and its associated nucleases in DNA interstrand crosslink repair Wouter S. Hoogenboom, Rick A.C.M. Boonen and Puck Knipscheer* Downloaded from https://academic.oup.com/nar/advance-article-abstract/doi/10.1093/nar/gky1276/5255686 by guest on 28 December 2018 Oncode Institute, Hubrecht Institute–KNAW and University Medical Center Utrecht, Utrecht, The Netherlands Received May 17, 2018; Revised December 11, 2018; Editorial Decision December 12, 2018; Accepted December 13, 2018 ABSTRACT the cancer predisposition syndrome Fanconi anemia (FA) that is caused by biallelic mutations in any one of the 22 A key step in the Fanconi anemia pathway of DNA currently known FA genes. Cells from FA patients are re- interstrand crosslink (ICL) repair is the ICL unhook- markably sensitive to ICL inducing agents, consistent with ing by dual endonucleolytic incisions. SLX4/FANCP the FA proteins being involved in the repair of DNA inter- is a large scaffold protein that plays a central role strand crosslinks (6,7). Indeed, it has been shown that ex- in ICL unhooking. It contains multiple domains that ogenous ICLs, for example caused by cisplatin, are repaired interact with many proteins including three different by the FA pathway (8). Although the source of the endoge- endonucleases and also acts in several other DNA nous ICL that requires the FA pathway for its repair is cur- repair pathways. While it is known that its interaction rently not known, genetic evidence points towards reactive with the endonuclease XPF-ERCC1 is required for its aldehydes (9–13).
    [Show full text]
  • A Mutation in the Putative MLH3 Endonuclease Domain Confers a Defect in Both Mismatch Repair and Meiosis in Saccharomyces Cerevisiae
    Copyright Ó 2008 by the Genetics Society of America DOI: 10.1534/genetics.108.086645 A Mutation in the Putative MLH3 Endonuclease Domain Confers a Defect in Both Mismatch Repair and Meiosis in Saccharomyces cerevisiae K. T. Nishant, Aaron J. Plys and Eric Alani1 Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853-2703 Manuscript received January 2, 2008 Accepted for publication March 20, 2008 ABSTRACT Interference-dependent crossing over in yeast and mammalian meioses involves the mismatch repair protein homologs MSH4-MSH5 and MLH1-MLH3. The MLH3 protein contains a highly conserved metal- binding motif DQHA(X)2E(X)4E that is found in a subset of MLH proteins predicted to have endonuclease activities (Kadyrov et al. 2006). Mutations within this motif in human PMS2 and Saccharomyces cerevisiae PMS1 disrupted the endonuclease and mismatch repair activities of MLH1-PMS2 and MLH1-PMS1, re- spectively (Kadyrov et al. 2006, 2007; Erdeniz et al. 2007). As a first step in determining whether such an activity is required during meiosis, we made mutations in the MLH3 putative endonuclease domain motif (-D523N, -E529K) and found that single and double mutations conferred mlh3-null-like defects with respect to meiotic spore viability and crossing over. Yeast two-hybrid and chromatography analyses showed that the interaction between MLH1 and mlh3-D523N was maintained, suggesting that the mlh3-D523N mutation did not disrupt the stability of MLH3. The mlh3-D523N mutant also displayed a mutator phenotype in vegetative growth that was similar to mlh3D. Overexpression of this allele conferred a dominant-negative phenotype with respect to mismatch repair.
    [Show full text]
  • A Genome-Wide Screen for Genes Affecting Spontaneous Direct-Repeat Recombination In
    bioRxiv preprint doi: https://doi.org/10.1101/2020.02.11.943795; this version posted February 11, 2020. 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. 1 A genome-wide screen for genes affecting spontaneous direct-repeat recombination in 2 Saccharomyces cerevisiae 3 4 5 Daniele Novarina*, Ridhdhi Desai†, Jessica A. Vaisica†, Jiongwen Ou†, Mohammed Bellaoui†,1, 6 Grant W. Brown†,2 and Michael Chang*,3 7 8 *European Research Institute for the Biology of Ageing, University of Groningen, University 9 Medical Center Groningen, 9713 AV Groningen, the Netherlands 10 †Department of Biochemistry and Donnelly Centre, University of Toronto, Toronto, ON M5S 11 3E1, Canada 12 13 1Current address: Genetics Unit, Faculty of Medicine and Pharmacy, University Mohammed 14 Premier, Oujda, Morocco 15 16 2Co-corresponding author: Department of Biochemistry and Donnelly Centre, University of 17 Toronto, 160 College Street, Toronto, ON M5S 3E1 Canada. E-mail: [email protected] 18 3Co-corresponding author: European Research Institute for the Biology of Ageing, University of 19 Groningen, University Medical Center Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, 20 the Netherlands. E-mail: [email protected] 21 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.11.943795; this version posted February 11, 2020. 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.
    [Show full text]
  • Role of Deubiquitinating Enzymes in DNA Repair Younghoon Kee University of South Florida, [email protected]
    University of South Florida Scholar Commons Cell Biology, Microbiology, and Molecular Biology Cell Biology, Microbiology, and Molecular Biology Faculty Publications 2-15-2016 Role of Deubiquitinating Enzymes in DNA Repair Younghoon Kee University of South Florida, [email protected] Tony T. Huang New York University School of Medicine Follow this and additional works at: http://scholarcommons.usf.edu/bcm_facpub Part of the Biology Commons, and the Cell and Developmental Biology Commons Scholar Commons Citation Kee, Younghoon and Huang, Tony T., "Role of Deubiquitinating Enzymes in DNA Repair" (2016). Cell Biology, Microbiology, and Molecular Biology Faculty Publications. 30. http://scholarcommons.usf.edu/bcm_facpub/30 This Article is brought to you for free and open access by the Cell Biology, Microbiology, and Molecular Biology at Scholar Commons. It has been accepted for inclusion in Cell Biology, Microbiology, and Molecular Biology Faculty Publications by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. crossmark MINIREVIEW Role of Deubiquitinating Enzymes in DNA Repair Younghoon Kee,a Tony T Huangb Department of Cell Biology, Microbiology, and Molecular Biology, College of Arts and Sciences, University of South Florida, Tampa, Florida, USAa; Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York, USAb Both proteolytic and nonproteolytic functions of ubiquitination are essential regulatory mechanisms for promoting DNA repair and the DNA damage response in mammalian cells. Deubiquitinating enzymes (DUBs) have emerged as key players in the main- tenance of genome stability. In this minireview, we discuss the recent findings on human DUBs that participate in genome main- tenance, with a focus on the role of DUBs in the modulation of DNA repair and DNA damage signaling.
    [Show full text]
  • Fanconi Anemia, Bloom Syndrome and Breast Cancer
    A multiprotein complex in DNA damage response network of Fanconi anemia, Bloom syndrome and Breast cancer Weidong Wang Lab of Genetics, NIA A Multi-protein Complex Connects Two Genomic Instability Diseases: Bloom Syndrome and Fanconi Anemia Bloom Syndrome . Genomic Instability: -sister-chromatid exchange . Cancer predisposition . Mutation in BLM, a RecQ DNA Helicase . BLM participates in: HR-dependent DSB repair Recovery of stalled replication forks . BLM works with Topo IIIa and RMI to Suppress crossover recombination Courtesy of Dr. Ian Hickson A Multi-protein Complex Connects Two Genomic Instability Diseases: Bloom Syndrome and Fanconi Anemia P I l o r t n o BLM IP kDa C HeLa BLAP 250 Nuclear Extract 200- BLM* FANCA* 116- TOPO IIIα* 97- BLAP 100 MLH1* BLM IP BLAP 75 * 66- RPA 70 IgG H 45- * 30- RPA32 IgG L 20- * 12- RPA14 Meetei et al. MCB 2003 A Multi-protein Complex Connects Two Genomic Instability Diseases: Bloom Syndrome and Fanconi Anemia P I A C N A F BLM IP HeLa FANCM= FAAP 250 BLAP 250 Nuclear Extract BLM* BLM* * FANCA* FANCA TOPO IIIα* TOPO IIIα* FAAP 100 BLAP 100 FANCB= FAAP 95 MLH1 FANCA IP BLM IP BLAP 75 BLAP 75 RPA70*/FANCG* RPA 70* FANCC*/FANCE* IgG H FANCL= FAAP 43 FANCF* RPA32* IgG L Meetei et al. MCB 2003 Meetei et al. Nat Genet. 2003, 2004, 2005 BRAFT-a Multisubunit Machine that Maintains Genome Stability and is defective in Fanconi anemia and Bloom syndrome BRAFT Super-complex Fanconi Anemia Bloom Syndrome Core Complex Complex 12 polypeptides 7 polypeptides FANCA BLM Helicase (HJ, fork, D-loop), fork FANCC regression, dHJ dissolution Topo IIIα Topoisomerase, FANCE dHJ dissolution FANCF BLAP75 RMI1 FANCG Stimulates dHJ dissolution.
    [Show full text]
  • HEREDITARY CANCER PANELS Part I
    Pathology and Laboratory Medicine Clinic Building, K6, Core Lab, E-655 2799 W. Grand Blvd. HEREDITARY CANCER PANELS Detroit, MI 48202 855.916.4DNA (4362) Part I- REQUISITION Required Patient Information Ordering Physician Information Name: _________________________________________________ Gender: M F Name: _____________________________________________________________ MRN: _________________________ DOB: _______MM / _______DD / _______YYYY Address: ___________________________________________________________ ICD10 Code(s): _________________/_________________/_________________ City: _______________________________ State: ________ Zip: __________ ICD-10 Codes are required for billing. When ordering tests for which reimbursement will be sought, order only those tests that are medically necessary for the diagnosis and treatment of the patient. Phone: _________________________ Fax: ___________________________ Billing & Collection Information NPI: _____________________________________ Patient Demographic/Billing/Insurance Form is required to be submitted with this form. Most genetic testing requires insurance prior authorization. Due to high insurance deductibles and member policy benefits, patients may elect to self-pay. Call for more information (855.916.4362) Bill Client or Institution Client Name: ______________________________________________________ Client Code/Number: _____________ Bill Insurance Prior authorization or reference number: __________________________________________ Patient Self-Pay Call for pricing and payment options Toll
    [Show full text]
  • Insights Into Regulation of Human RAD51 Nucleoprotein Filament Activity During
    Insights into Regulation of Human RAD51 Nucleoprotein Filament Activity During Homologous Recombination Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Ravindra Bandara Amunugama, B.S. Biophysics Graduate Program The Ohio State University 2011 Dissertation Committee: Richard Fishel PhD, Advisor Jeffrey Parvin MD PhD Charles Bell PhD Michael Poirier PhD Copyright by Ravindra Bandara Amunugama 2011 ABSTRACT Homologous recombination (HR) is a mechanistically conserved pathway that occurs during meiosis and following the formation of DNA double strand breaks (DSBs) induced by exogenous stresses such as ionization radiation. HR is also involved in restoring replication when replication forks have stalled or collapsed. Defective recombination machinery leads to chromosomal instability and predisposition to tumorigenesis. However, unregulated HR repair system also leads to similar outcomes. Fortunately, eukaryotes have evolved elegant HR repair machinery with multiple mediators and regulatory inputs that largely ensures an appropriate outcome. A fundamental step in HR is the homology search and strand exchange catalyzed by the RAD51 recombinase. This process requires the formation of a nucleoprotein filament (NPF) on single-strand DNA (ssDNA). In Chapter 2 of this dissertation I describe work on identification of two residues of human RAD51 (HsRAD51) subunit interface, F129 in the Walker A box and H294 of the L2 ssDNA binding region that are essential residues for salt-induced recombinase activity. Mutation of F129 or H294 leads to loss or reduced DNA induced ATPase activity and formation of a non-functional NPF that eliminates recombinase activity. DNA binding studies indicate that these residues may be essential for sensing the ATP nucleotide for a functional NPF formation.
    [Show full text]
  • Slx1–Slx4 Is a Second Structure-Specific Endonuclease Functionally Redundant with Sgs1–Top3
    Downloaded from genesdev.cshlp.org on September 30, 2021 - Published by Cold Spring Harbor Laboratory Press Slx1–Slx4 is a second structure-specific endonuclease functionally redundant with Sgs1–Top3 William M. Fricke and Steven J. Brill1 Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854, USA The RecQ DNA helicases human BLM and yeast Sgs1 interact with DNA topoisomerase III and are thought to act on stalled replication forks to maintain genome stability. To gain insight into this mechanism, we previously identified SLX1 and SLX4 as genes that are required for viability and for completion of rDNA replication in the absence of SGS1–TOP3. Here we show that SLX1 and SLX4 encode a heteromeric structure-specific endonuclease. The Slx1–Slx4 nuclease is active on branched DNA substrates, particularly simple-Y, 5؅-flap, or replication forkstructures. It cleaves the strand bearing the 5 ؅ nonhomologous arm at the branch junction and generates ligatable nicked products from 5؅-flap or replication forksubstrates. Slx1 is the founding member of a family of proteins with a predicted URI nuclease domain and PHD-type zinc finger. This subunit displays weakstructure-specific endonuclease activity on its own, is stimulated 500-fold by Slx4, and requires the PHD finger for activity in vitro and in vivo. Both subunits are required in vivo for resistance to DNA damage by methylmethane sulfonate (MMS). We propose that Sgs1–Top3 acts at the termination of rDNA replication to decatenate stalled forks, and, in its absence, Slx1–Slx4 cleaves these stalled forks. [Keywords: Replication restart; RecQ helicase; DNA topoisomerase; endonuclease; Mus81–Mms4] Received April 21, 2003; revised version accepted May 22, 2003.
    [Show full text]
  • Linking the Multiple Functions of Xpf-Ercc1 Endonuclease in Dna Repair to Health Outcomes: Cancer and Aging
    LINKING THE MULTIPLE FUNCTIONS OF XPF-ERCC1 ENDONUCLEASE IN DNA REPAIR TO HEALTH OUTCOMES: CANCER AND AGING by Advaitha Madireddy B. Tech, SRM University, India, 2008 Submitted to the Graduate Faculty of Graduate School of Public Health in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2012 UNIVERSITY OF PITTSBURGH Graduate School of Public Health This dissertation was presented by Advaitha Madireddy It was defended on June 25th, 2012 and approved by Candace M. Kammerer, PhD, Assistant Professor, Human Genetics, Graduate School of Public Health, University of Pittsburgh Susanne M. Gollin, PhD, Professor, Human Genetics, Graduate School of Public Health, University of Pittsburgh Patricia L. Opresko, PhD, Assistant Professor, Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh Dissertation Advisor: Laura J. Niedernhofer, MD, PhD, Associate Professor, Microbiology and Molecular Genetics, School of Medicine, University of Pitttsburgh ii Copyright © by Advaitha Madireddy 2012 iii LINKING THE MULTIPLE FUNCTIONS OF XPF-ERCC1 ENDONUCLEASE IN DNA REPAIR TO HEALTH OUTCOMES: CANCER AND AGING Advaitha Madireddy, PhD University of Pittsburgh, 2012 XPF-ERCC1 is a structure specific endonuclease in which the XPF subunit is involved in nucleolytic activity and the ERCC1 subunit is involved in DNA binding. They are essential for multiple genome maintenance mechanisms which include the repair of bulky DNA monoadducts via nucleotide excision repair (NER) and also the repair of DNA interstrand crosslinks. In humans, the deficiency of XPF-ERCC1 results in two major syndromes: Xeroderma pigmentosum (XP), characterized by predisposition to skin cancer and XFE, characterized by symptoms of premature aging.
    [Show full text]