MutY ADENINE DNA GLYCOSYLASE

1
Mut-Y ADENINE DNA GLYCOSYLASE

• SHRUTI PADHEE

2
What are DNA GLYCOSYLASES?

• They are a class of enzymes which are responsible
  for the Base-excision repair of DNA.
• Base excision repair is the mechanism by which
  nucleotide residues in DNA with chemically
  altered nitrogen bases can be removed and
  replaced.
• The sugar phosphate backbone is intact.
• Found in every cell.
• They recognize common DNA lesions and remove the
  affected base by cleaving the N-glycosyl bond and
  they generate an abasic or AP site
  (apurinic/apyrimidinic).
• These AP sites are recognised by AP endonuclease
  enzymes which complete the rest of the repair.

3
FORMATION OF THE ABASIC SITE
4
General Mechanism of action of DNA glycosylase

• Recognition of damaged base.
• Cleavage between deoxy -ribose backbone and base.
• AP endonuclease cleaves phosphodiester backbone
  near AP site.
• DNA polymerase initiates repair synthesis from
  the free 3 OH (with its 5 to 3exonuclease
  activity) replaces it with undamaged DNA.
• The nick remaining is sealed by the DNA ligase.

5
Interesting facts about DNA Glycosylases

• They locate and excise the damaged DNA
  nucleobases in a 1 million fold excess of
  undamaged DNA.
• When the enzyme encounters an 8-oxo guanine it
  flips the base out of the DNA helix into its
  active site in order to remove it.
• The enzyme flips the base into a gateway pocket
  that regulates entry to the active site, allowing
  the undamaged guanines that enter to return to
  the helix unharmed.

6
8- oxo guanine (oxoG)

• It is a genotoxic product .
• Formed due to the oxidation of guanine in aerobic
  organisms.

guanine
Oxidation at position 8
7
Pathway for oxidation and repair of guanine in DNA
Normal DNA
8-oxo guanine. c complex
Abasic site/AP site
8
Major goals

• To work out a strategy to catch the DNA
  glycosylases in the act of searching the haystack
  for damaged DNA nucleobases.
• To develop a powerful cross linking system to
  help crystallize the otherwise un-crystallizable
  complexes of DNA repair proteins bound to short
  stretches of DNA.

9
Developed method

• A powerful crosslinking system was developed in
  which thiol groups are strategically installed in
  both the protein and the DNA substrate .
• They are then tethered via a disulphide bond
  ,making it possible to isolate the normally
  fleeting protein-DNA complexes.

10
Crystallized full-length MutY (mol wt 41000)
interacting with DNA

• The MutY glycosylase consists of 3 major regions
• Catalytic domain
• 6- helix barrel module
• C-terminal domain

OXO-G
?-STRANDS
DNA
ADENINE
HELICES
90 deg rotated towards the reader
11

• 1) Catalytic domain It has a helix hairpin
  helix element, a gly/pro rich loop,
• and a catalytically essential aspartate residue (
  HhH -GPD motif).
• It bears resemblence to endonuclease III
  including the 4 Fe-4S cluster .
• This domain interacts with the adenine residue.
• The substrate adenine is completely extruded
  from the DNA helix and is
• Inserted into an extra helical pocket in the
  catalytic domain , a feature
• shared by all known DNA Glycosylases that act on
  single base lesions.
• 2) 6- helix barrel module It directly contacts
  the backbone of complementary
• Oxo G containing strand.
• The DNA substrate is bent by 55 deg (similar to
  the endonuclease III)
• The bend is localized to the lesion and the
  normal unbent B form of
• DNA projects from either side.
• 3) C- Terminal domain It is thought to be
  responsible for the OxoG recognition
• as the removal of this region leads to the loss
  of discrimination between the
• A.oxoG and the A. G .
• This domain is linked to the catalytic domain by
  disordered linker of 10 amino
• acids (rich in lys -228,230,231,235) that
  traverses major groove near the lesion.

12
Resemblence of Mut Y with MutT and the Endo
IIIDNA complex

• Mut T sanitizes the the
• Nucleotide precursor pool by
• hydrolysing oxo-dGTP to
• Oxo-d GMP and inorganic
• Phosphate

13
Molecular surface representation of enzyme bound
to DNA

• The enzyme encircles the DNA duplex burying 1530
  angstrom square of DNA surface area.
• MutY deeply penetrates DNA helix ,interrupts
  helical stacking on both strands .
• It enforces a sharp bend in the DNA and
  extrahelical extrusion of adenine.
• The oxo-G lesion lies in the DNA helix.
• The lesion recognition causes no conformational
  change in the catalytic domain of the enzyme.

14
Ball and stick representation of the actual
interaction

• The oxo G has a an anti glycosidic bond
  conformation when bound to MutY and is in syn
  conformation when bound to the adenine.
• The oxo-G swivels 180 deg to drive the extrusion
  of adenine out of the helix.
• The aromatic ring of Tyr 88 from the enzyme
  intercalates into 5side of oxo-Guanine.
• Now the Gln 48 inserts into the helical space
  vacated by the adenine to form p-stacking
  interactions under the 3-neighbouring base and
  simultaneous hydrogen bonds with the 3
  phosphate of adenine.

15
Various interactions stabilizing the oxo-G

• (1. )The watson and crick face of oxo-G is
  contacted by
• Gln 48(amide carbonyl)
• Thr 49(hydroxyl )
• (2.) The minor groove face of
• oxo-G makes its contact with
• Leu 86(hydrogen bonding)
• (3.) The hoogstein face of oxo-G is stabilized by
  
• Ser 308(which is inturn stabilized by the tyr88
  hydroxyl)

16
Catalytic strategy used by Mut Y to remove the
adenine

• DNA glycosylases act by nucleophillic SN-1 attack
  by water in discrete steps.
• Two water molecules and the Glu43 are involved in
  the reaction where Glu43 is
• responsible to lower the transition energy
  during base excision.
• The N7 of adenine is replaced wth the c7-h yeild
  a substrate product that cannot be
• Cleaved and binds to the MutY .
• All this is possible only when the adenine is in
  syn configuration.

17
Conclusions drawn

• The results suggests the molecular basis for
  MutYs preferential recognition of oxo G versus
  thymine even though T.A is 100000- 1000000 fold
  greater than that of oxoG.A.
• The oxo-G recognition mode of Mut Y is different
  than that of MutT though they have similar C
  terminal domains .
• The enzyme shows greater than a 6-fold preference
  for A.oxoG over A.G in which Ser 308 (binds to o8
  of oxo-G not possible with G) primarily
  responsible for discriminating between oxoG and
  guanine.

18
Relevance to the field of science

• The disulphide cross linking technique is used
• To reveal how a bacterial DNA repair enzyme
  (Mut-Y) uses a hydrophobic probe residue to
  examine the intact DNA helix for 8-oxoguanine.
• To figure out how the human version of this
  enzyme (hMYH) ensures that only the 8-oxoguanine
  is flipped into the active site and removed.
• In mechanistic crystallography.
• To capture mechanistically revealing snapshots
  of voltage gated ion channels.

19
Methods used

• MutY preparation It is prepared from Bacillus
  stearothermophilus.
• The gene is cloned into the PET 228 expression
  vector.
• The Asp144Asn and Pro164cys mutation is generated
  using mutagenesis kit .
• The cells are lysed by sonification to remove the
  enzyme.
• DNA ,complex formation and disulphide trapping
  The DNA oligomers 5-TGTCCAXGTCT-3(X-ABASIC
  SITE),5-AAGACYTGGAC-3(Y-OXOG) were synthesized.
• A-stands for the modified nucleoside introduced
  for disulphide linking.

20

• Crystallization

• The complexes were crystallized by hanging drop
  method at room temperature in
• 100mM Tris ,14PEG,500mM calcium acetate and 5mM
  ß- ME.
• Crystals appeared after several days and were
  allowed to grow for several weeks.
• Frozen in liquid nitrogen for the x-ray data
  collection.

21
Summary

• MutY is a adenine Glycosylase enzyme.
• Mut Y is very selective for oxo G .A complex.
• The adenine is removed by nucleophillic attack by
  water.
• The crystalline structure was studied by making
  use of disulphide crosslinking.

22
References

• J.christopher Fromme1,Anirban banerjee2,Susan
  J.Huang 1 GregoryL.Verdine 1,2.(Department of
  molecular and Celllular biology and department
  of chemistry and chemical biology,Harvard
  university,cambridge,,Massachusetts 02138)
• 2007 ACS national award winners, CEN ,pg 32, feb
  5 ,2007
• Principles of Biochemistry, Lehninger, David
  M.Nelson ,Michael M. Cox ,chapter 25.

23
Acknowledgements

• Dr. Jon A. Friesen

24
ANY QUESTIONS

• ?