Sandbox Reserved 312

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This Sandbox is Reserved from January 10, 2010, through April 10, 2011 for use in BCMB 307-Proteins course taught by Andrea Gorrell at the University of Northern British Columbia, Prince George, BC, Canada.
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Contents

WRN exonuclease

PDB ID 2fbv

Drag the structure with the mouse to rotate
2fbv, resolution 2.40Å ()
Ligands:
Gene: WRN, RECQ3, RECQL2 (Homo sapiens)
Related: 2fbt, 2fbx, 2fby, 2fc0
Resources: FirstGlance, OCA, RCSB, PDBsum
Coordinates: save as pdb, mmCIF, xml


Introduction

Werner (WRN) protein is a member of the human RecQDNA helicases.[1] This enzyme has a helicases function (unwinding double-stranded DNA helix in the 3'-5' direction) and a exonuclease activity (degrading), which allows for deletion of mutation and proofreading.[1] The WRN has a functional exonuclease domain located on the N-terminus.[1] WRN protein protects human from cancer; as well as, premature aging. [2]

Structure

The WRN protein is built from several individual WRN copies. The exonuclease region of the protein is located at the N-terminal end with 171 amino acids.[3] They form a hexamer around the end of the strand when exposed to double strand DNA, which is essential to DNA repair of the WRN.[1]

The HRDC domain has 80 amino acids at the C-terminal end.[4] There are five helicases connected by turns and hydrophobic loops. These hydrobopic loops are needed for for packing the domain into a bundle. This domain contributes in the interaction with the double stranded DNA.[4]

The catalysis the exonuclease activity by having the , Mn2+ or Mg2+ complex. Mg2+ are more commonly cofactors in the cell; however, Mn2+ supports higher catalytic activity. This active site contains Glu84, Tyr212, Ala188, Glu192, Asp216, Asp82, Tyr54 and Asp143.[1] When bond to the active site the two metals have a distance of 3.7 Å separating them when bound to the active site.[1] The inner Mn2+ is directly coordinated with Asp82, Glu84 and Asp216.[1] The outer metal ion binds with a ligatures side chain Asp82 to form the bridge between the two metal ions.[1] Asp143 interacts indirectly with the outer metal ion by two water molecules. [1] The binding of the two metal ions do not change conformation of the WRN protein but the correct bond is vital for the activity of the WRN exonuclease.[1]

Besides the two Mn cations association sites this protein has two conserved ATP binding sites. This ATP binding and hydolysis is depenent on Mn binding.[5] Therefore, WRN is a enzyme with both Mn and ATP-dependent DNA-helicase activity with a 3'-5' exonuclease activity towards double-stranded DNA.[5] This provides energy for DNA helicase, breaking hydrogen bonds between bases of double stranded DNA, which unwinds the helix. A six membered WRN ring is formed to fit around the DNA helix. The exonuclease active site and Mn2+ point inward towards the DNA. [5]

Function

This protein plays a major role in DNA metabolic pathway, which perform DNA repair and genome stability. The protein is a 3`-5`helicase which indicates that it cannot be used to transcription and replication (5`-3`). WRN has activity of ATPase, helicases and exonuclease. [6] WRN exonuclease functions on different structures of a DNA substrate.[6] The metal ions Mn2+ are needed to stimulate the exonuclease active of the WRN. However, WRN exonuclease can be blocked by oxidatively induced base lesion (damaged by disease tissue).[7] The homodimer protein Ku binds to the broken double-stranded DNA, stimulating the WRN exonuclease to bypass the lesion. However, in non-digested strands the process is blocked by the lesion.[7]

WRN are found in tissues, mainly in the pancreas and testis. In cells, WRN is found in the nucleus and nucleolar regions.[7]

DNA attached to WRN-exo
DNA attached to WRN-exo

Clinical significance

The build up of oxidatively induced base in DNA the gene coding for WRN is mutated a autosomal recessive disorder causing rapid aging(osteoporosis, atherosclerosis and cancer). This is known as Werner syndrome (WS) which appears at puberty.[8]

Mutation in the WRN gene occurs int the HRDC domain.[4] This domain actives the DNA association with the helicase; as a result, a mutation in this region blocks DNA repair mechanism to occur.[4] As DNA cell damage accumulates cell death occurs losing WRN protein function, leading to premature aging.[4]

Reference

  1. ↑ 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 Perry JJ, Yannone SM, Holden LG, Hitomi C, Asaithamby A, Han S, Cooper PK, Chen DJ, Tainer JA. WRN exonuclease structure and molecular mechanism imply an editing role in DNA end processing. Nat Struct Mol Biol. 2006 May;13(5):414-22. Epub 2006 Apr 23. PMID:16622405 doi:http://dx.doi.org/10.1038/nsmb1088
  2. ↑ Choudhary S, Sommers JA, Brosh RM Jr. Biochemical and kinetic characterization of the DNA helicase and exonuclease activities of werner syndrome protein. J Biol Chem. 2004 Aug 13;279(33):34603-13. Epub 2004 Jun 8. PMID:15187093 doi:10.1074/jbc.M401901200
  3. ↑ Bacolla A, Wang G, Jain A, Chuzhanova NA, Cer RZ, Collins JR, Cooper DN, Bohr VA, Vasquez KM. Non-B DNA-forming Sequences and WRN Deficiency Independently Increase the Frequency of Base Substitution in Human Cells. J Biol Chem. 2011 Mar 25;286(12):10017-26. Epub 2011 Feb 1. PMID:21285356 doi:10.1074/jbc.M110.176636
  4. ↑ 4.0 4.1 4.2 4.3 4.4 Kitano K, Yoshihara N, Hakoshima T. Crystal structure of the HRDC domain of human Werner syndrome protein, WRN. J Biol Chem. 2007 Jan 26;282(4):2717-28. Epub 2006 Dec 4. PMID:17148451 doi:10.1074/jbc.M610142200
  5. ↑ 5.0 5.1 5.2 Hu JS, Feng H, Zeng W, Lin GX, Xi XG. Solution structure of a multifunctional DNA- and protein-binding motif of human Werner syndrome protein. Proc Natl Acad Sci U S A. 2005 Dec 20;102(51):18379-84. Epub 2005 Dec 9. PMID:16339893
  6. ↑ 6.0 6.1 Brosh RM Jr, Opresko PL, Bohr VA. Enzymatic mechanism of the WRN helicase/nuclease. Methods Enzymol. 2006;409:52-85. PMID:16793395 doi:10.1016/S0076-6879(05)09004-X
  7. ↑ 7.0 7.1 7.2 Li B, Comai L. Requirements for the nucleolytic processing of DNA ends by the Werner syndrome protein-Ku70/80 complex. J Biol Chem. 2001 Mar 30;276(13):9896-902. Epub 2001 Jan 4. PMID:11152456 doi:10.1074/jbc.M008575200
  8. ↑ Opresko PL, Laine JP, Brosh RM Jr, Seidman MM, Bohr VA. Coordinate action of the helicase and 3' to 5' exonuclease of Werner syndrome protein. J Biol Chem. 2001 Nov 30;276(48):44677-87. Epub 2001 Sep 25. PMID:11572872 doi:10.1074/jbc.M107548200
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