BIOL368/F14:Isabel Gonzaga Week 6

STAR Biochem DNA Glycosylase

Methods and Results

Method taken from DNA Glycosylase Exercise [StarBiochem] was downloaded and ran using a Java applet. I hit start, then selected: Samples > Select from Samples. Within the Amino Acid/Proteins > Protein tab, select “DNA glycosylase hOGG1 w/ DNA – H. sapiens (1EBM)”

1. The hOGG1 structure contains both DNA and protein. Can you differentiate between the DNA and protein components? How did you distinguish the DNA from the protein?
   • The DNA Sequence forms an alpha helix on the side of the page. This structure is very easily recognizable. because of the planar base stacking. the protein complex contains many side chains and has a repetitive pattern of peptide bonds (featuring nitrogen and carbon atoms)
2. hOGG1 contains multiple sulfur atoms.Identify the name and sequence number of one of the amino acids in the structure that contains a sulfur atom.
   • • Answer: Cysteine, position 146. This is located in the side chain of the amino acid.
     • Procedure:
       • I pointed to a yellow (sulfur) Atom in structure. A small box appeared on top of the mouse cursor indicating the name of the amino acid and its position in the amino acid sequence
       • From the top menu, I selected: View> View Specific Regions / Set Center of Rotation. I selected the CYS at position 146. I moved the VDW Radius slider all the way to the left (1 Van der Waals radii) and zoomed in. Unchecking the back bone box (leaving side chain), I saw that it must be a part of the side chain.

1. Next, I explored the primary structure of the hOGG1 protein. The hOGG1 protein consists of 325 amino acids. List the 13 amino acids numbered 105 through 117 in order.
   • Answer: Threonine, Leucine, Alanine, Glutamine, Leucine, Tyrosine, Histidine, Histidine, Tryptophan, Glycine, Serine, Valine, Aspartate
   • Procedure: I reset the structure. Hit Protein -> Primary tab and found the 3 letter codes for amino acids 105-117.

1. Within a protein chain, amino acids form local structures called secondary structures (Reference page).
   • a) Explore the secondary structures found in hOGG1. Are helices, sheets or coils present in hOGG1? Describe the color that represents the secondary structures you observe.
     • Answer:
       • Alpha Helices: Magenta
       • Beta Sheets: Yellow
       • Random Coil: Blue
     • Procedure: I clicked on the secondary tab and checked the box beside the desired structure (e.g. helices, sheets and coils) and moved the structure size slider to the right to increase the size, one by one.
   • b) Amino acids 105 through 117 fold into one of the secondary structures. Which secondary structure do they fold into?
     • Answer: Alpha Helix
     • Procedure: In the View Specific Regions window, I went to the Protein - Secondary tab and clicked the All button. In the amino acid sequence window, I selected 105-117 and moved the VDQ radius slider to the left to show only the selected amino acids in the viewer.

1. Then I explored the relationship between DNA glycosylase’s structure and one of the several types of amino acids that contribute to hOGG1’s overall shape, its tertiary structure (Reference page).
   • a) Negatively charged amino acids are hydrophilic (Reference page). Are the negatively charged amino acids located on the inside (buried) or outside (exposed) of this protein? What does that suggest about the cellular environment surrounding this protein, is it hydrophobic or hydrophilic? Explain your answer.
     • Answer: Negatively charged atoms are on the outside of the protein. This means that the cellular environment must be hydrophilic, because it interacts with the polar, charged atoms.
     • Procedure: I reset the structure. Then I clicked on the tertiary tab, and moved the atoms size slider to the left to decrease size for all atoms in the protein. I clicked on the negatively charged/acidic button and moved the atom size slider to the right to increase the atom size for the negatively charged/acidic amino acids.

1. Then I explored how DNA glycosylase interacts with DNA to recognize the damaged DNA base within its sequence. DNA is composed of four bases: Adenine (A), Thymine (T), Cytosine (C) and Guanine (G). In this particular structure, the hOGG1 protein is bound to a segment of DNA that contains an oxidized guanine. .
   • FIrst, I made the DNA more visible
     • I reset structure, then clicked on the Nucleic Acids tab to move the Atoms Size slider to the right (35%), increasing the size of the DNA segment.
     • In the Protein - Primary tab, I moved the Atoms Size slider completely to the left and the Bonds Translucency slider to the right (95%) to minimize the appearance of all the amino acids in the hOGG1 protein.
   • Second, I looked at how DNA bases are oriented within the double helix. Each base is attached to a sugar and phosphate backbone to form a complete nucleotide. Within the double helix, a base within one strand pairs with another base from the opposite strand by hydrogen bonding forming a base pair: Adenine (A) base pairs with Thymine (T) and Cytosine (C) base pairs with Guanine (G).
     • Nucleic Acids tab: unchecked phosphates and sugars. I moved the Atoms Size slider to the right (70%) to increase the size of the bases while leaving the size of the phosphates and sugar nucleotide components intact. This allowed me to count the bases more easily. Then, I clicked on the Non-Peptide tab. I moved the Atoms Size slider completely to the right to increase the size of the atoms in the oxidized guanine ([8OG]25).
   • Questions
     • a) How many DNA base pairs can you count within this double helix? How many bases are unpaired?
       • Answer: 14
     • b) How many DNA bases are unpaired (not paired to its partner on the other strand)?
       • 2 unpaired bases
     • c) Is the oxidized guanine base paired or unpaired? Describe the position of the oxidized guanine with respect to the hOGG1 protein and the double helix. What does this suggest about the mechanism that hOGG1 uses to identify damaged DNA bases?
       • The oxidized guanine base is unpaired. the base is positioned away from the double stranded DNA helix. This suggests that the hOGG1 identifies damaged bases based on their orientation relative to the rest of the molecule. If it doesn't fit exactly or sticks out or is oriented away, then the protein will come and repair the damaged site.

1. Certain amino acids within hOGG1 form contacts with the DNA and are able to recognize if a guanine base has been damaged by oxidation. Where are you more likely to find the amino acids that recognize damaged guanine bases within hOGG1, in Helix 1 or Helix 16? Explain why.
   • Answers:
     • Helix 16 recognized more damaged guanine bases. This is 310-324. The sequences are as follows:
       • Helix 16: Tyrosine, Alanine, Glycine, Tryptophan, Alanine, Glutamine, Alanine, Valine, Leucine, Phenylalanine, Serine, Alanine,Aspartate, Leucine, Arginine
       • Helix 1: Threonine, Proline, Alanine, Leucine, Tryptophan
     • The damaged guanine base is oxidized, and now has a carbonyl on the nitrogenous ring. Helix 16 is better able to recognize this, because in protein folding, it is located closer. Additionally, helix 16 has more polar side chains that directly interact with the oxidize guanine. This includes Glutamine. The phenylalanine in the side chain is aromatic and is attracted to the aromatic nitrogenous rings in the guanine base, as it it 'stacks' on top of it due to the overlapping pi bonds. The 15 amino acids composes a larger, bulkier chain. This is thus effective in recognizing damaged DNA, particularly if the nucleotide base is sticking outwards on the side of the helices. Overall, the structure and amino acid side chain activity allows Helix 16 to be more efficient in determining bases damaged by oxidation.
   • Procedure:
     • In the Protein - Secondary tab, I clicked the All button. In the Sequence Window, I selected the amino acids within Helix 1 and increased the size of Helix 1 by moving the Secondary Structures size slider to the right. I repeated this for Helix 16.
     • I then examined if the side chains in Helix 1 or Helix 16 contacted the oxidized guanine ([8OG]25). I clicked on the Protein - Primary tab, Selected the amino acids that make up Helix 1 in the Sequence Window, Unchecked backbone, and moved the Atoms Size slider to the right to visualize the side chains of the amino acids Helix 1. Then I repeated this for Helix 16

helix16sidegroups.png

1. hOGG1 is able to distinguish very efficiently between guanine and its oxidized counterpart, 8-oxoguanine. This represents a formidable task given that the oxidized nucleobase, 8-oxoguanine, differs by only two atoms from its normal counterpart, guanine (positions 7 and 8). The following structures illustrate hOGG1 bound to either an 8-oxoguanine or a guanine nucleobase: “1YQR” (8-oxoguanine) and “1YQK” (guanine). I compared these two structures to understand how hOGG1 can effectively distinguish between 8-oxoguanine and guanine, using the diagram under the [section] for details.
   • Procedure
     • In the top menu, I selected 'Samples' > 'Select from Samples'. Within the Amino Acid/Proteins - Protein tab, I selected “DNA glycosylase hOGG1 w/ DNA containing oxoG - H. sapiens (1YQR)”. “1YQR” is the four character ID for this particular structure.
       • I repeated these steps to open the “DNA glycosylase hOGG1 w/ DNA containing G - H. sapiens (1YQK)” structure. Glycine 42 in hOGG1 directly interacts with the base portion of 8-oxoguanine. This interaction is crucial for detection of oxidative damage of guanine bases.
       • In the Protein - Primary tab move the Atoms Size slider completely to the left and the Bonds Translucency slider to completely to the right to minimize the appearance of all the amino acids in the hOGG1 protein.Click on the Nucleic Acids tab. Click the oxidized guanine ([8OG]23:C) and increase its size by moving the Atoms Size slider completely to the right. In the Protein  Primary tab, click glycine 42 and increase its size by moving the Atoms Size slider completely to the right
   • Questions
     • a) Which atom in the base portion of 8-oxoguanine is directly contacting glycine 42? Draw this interaction (use the chemical structures within this question and the amino acids structures found in the Reference page).
       • The hydrogen from 8-oxoguanine
     • b) What does the interaction between 8-oxoguanine and glycine 42 indicate about how hOGG1 differentiates between 8-oxoguanine and guanine?
       • The carboxyl terminal from glycine hydrogen bonds with the a hydrogen on the nitrogenous base of the 8-oxoguanine. In guanine, there is a lone pair on the nitrogen, so it is unable to react I'm this way. Glycine is likely relevant here because it is the smallest structure, and thus is able to interact at this level with little hinderance.
   • c) Compare the general location and orientation of guanine and glycine 42 in the “1YQK” structure with that of 8-oxoguanine and glycine 42 in the “1YQR” structure. How do the location and orientation of these two nucleobases and glycine 42 differ in these structures? Explain how this comparison adds to your understanding of hOGG1’s ability to discriminate between 8-oxoguanine and guanine.
     • • Glycine in both structures are generally located in the same area. However, glycine in the 1YQK structure is directly hydrogen bonding with the 8-oxoguanine. The carboxyl group on the amino acid bonds to the hydrogen on the nitrogenous ring. The orientation is thus different for the 1YQR structure, as the hydrogen on the glycine is more oriented towards the oxygen on the nitrogenous ring. This demonstrates how hOGG1 is able to shif it's folding in higher levels dependent on the ionizability of mutated DNA molecules. Thus, amino acid structure and characteristics become very important in determining reactivity and recognizing key factors.

1. • • Reset the “1YQR” structure by clicking on Reset  Reset structure in the top menu.
     • In the Protein  Primary tab move the Atoms Size slider completely to the left and the Bonds Translucency slider to completely to the right (95%) to minimize the appearance of all the amino acids in the hOGG1 protein.
     • Click on the Nucleic Acids tab. Click the oxidized guanine ([8OG]23:C) and increase its size by moving the Atoms Size slider completely to the right.
     • In the Protein  Primary tab, click glycine 42 and increase its size by moving the Atoms Size slider completely to the right.
     • Repeat these steps with the “1YQK” structure, but instead of increasing the size of the oxidized guanine, increase the size of guanine at position 23.
2. One intriguing question about hOGG1 and other DNA glycosylases is whether these enzymes search for DNA oxidation by extruding each base from DNA and presenting it for inspection to the active site or by recognizing DNA oxidation sites without extruding each base. It is known that the base pairing between the 8-oxoguanine nucleobase and cytosine (oxoG:C) does not result in a conformational change in the DNA double helix. Therefore, it seems unlikely that DNA glycosylases would be able to correctly detect the presence of an 8-oxoguanine nucleobase without extruding each base. Yet, DNA glycosylases need to survey approximately 6x109 base pairs in the human genome, making the extrusion of each base energetically prohibitive and extremely time-consuming. Crystal structure evidence of a bacterial homolog of hOGG1, called MutM, suggests that a specific amino acid, phenylalanine 114, in MutM intercalates into the DNA helix at each nucleotide position as DNA glycosylase travels along the DNA - if you are curious, this structure is found within Samples and it is called “DNA glycosylase MutM w/ DNA - G. stearothermophilus (2F5O)”. It has been hypothesized that phenylalanine 114 acts as a sensor to detect the presence of 8-oxoguanine nucleobases. To explain how intercalation of phenylalanine at the site of an oxoG:C would disrupt base pairing and promote extrusion of the 8-oxoguanine nucleobase, but not of A:T and G:C base pairs, it has been suggested that intercalation of phenylalanine 114 at the site of an oxoG:C base pair may sufficiently destabilize base pairing because oxoG:C base pairs are less stable than A:T and G:C pairs. Propose an experiment to test whether phenylalanine 114 plays a role in the detection and repair of 8-oxoguanine nucleases