BIOL368/F14:Nicole Anguiano Week 6

Star Biochem DNA Glycosylase Exercise

Methods and Results

• First, I downloaded Star BioChem, and began following the instructions from the DNA Glycosylase Exercise worksheet.
• I selected the “DNA glycosylase hOGG1 w/ DNA – H. sapiens (1EBM)” protein from the Samples > Select from Samples menu. I changed the size of the atoms from 20 to 100 to view the protein as a space-filling model.

Figure 1: The space-filling model of DNA Glycosylase. Note the smaller, less-visible DNA displayed as a ball and stick model.

• Next, I pointed to a sulfur atom in the hOGG1 structure to obtain information about it.

Figure 2: This sulfur atom is part of cysteine at position 241.

• I selected View > View Specific Regions / Set Center of Rotation, then selected the amino acid in Figure 2. I moved the VDW Radius slider to 1 and unchecked the side-chain box. I observed that the sulfur atom disappeared. I checked the side-chain box and unchecked the backbone box. The sulfur atom remained. From there, I deduced that the Sulfur atom is a part of the side-chain.

Figure 3: The position of the sulfur atom at amino acid 241. As the "backbone" box is unchecked and the atom is still visible, it can be deduced that it is in the side-chain.

• I reset the structure by selecting Reset > Reset structure in the top menu, then scrolled down and found amino acids 105-117.

Number	Amino Acid
105	Threonine (Thr)
106	Leucine (Leu)
107	Alanine (Ala)
108	Glutamine (Gln)
109	Leucine (Leu)
110	Tyrosine (Tyr)
111	Histidine (His)
112	Histidine (His)
113	Tryptophan (Trp)
114	Glycine (Gly)
115	Serine (Ser)
116	Valine (Val)
117	Aspartic Acid (Asp)

• Table 1: The identity of amino acids number 105 to 117.
• I clicked on the secondary tab and selected "all" to view all of the different structures (helices, coils, and sheets). I moved the Structures Size slider to the right to make them larger. I selected View > View Specific Regions / Set Center of Rotation, and selected amino acids 105 to 117 by clicking on amino acid 105, holding Shift, then clicking on amino acid 117. I moved the VDM Radius slider to 1 so that I could just see the selected amino acids.

Figure 4: The alpha helix formed by amino acids 105 to 117.

• I reset the structure by selecting Reset > Reset structure in the top menu. I selected the Tertiary tab and moved the atom slider to the left to make all the atoms as small as possible. I then selected the negatively charged/acidic radio button. I moved the Atoms Size slider to the right to make the acidic amino acids as large as possible.

Figure 5: An image displaying the locations of the negatively charged/acidic amino acids in the hOGG1 structure.

• I reset the structure by selecting Reset > Reset structure in the top menu. I selected the Nucleic Acids tab and moved the Atoms Size slider to 35. I then selected the Protein > Primary tab and moved the Atoms Size slider to 0 and the Bonds Translucency slider to 95. I selected the Nucleic Acids tab again and unchecked the phosphates checkbox and the sugar atoms checkbox. I moved the Atoms Size slider to 70. I clicked on the Non-Peptide tab and moved the Atoms Size slider to 100 so that I could fully observe the oxidized guanine.
• Without resetting the structure, I selected the Protein > Secondary tab and selected "all". Next, I selected the amino acids within helix 1 by clicking on its first amino acid, holding shift, and clicking the last amino acid in the helix. I increased the Secondary Structures Size slider to 100. I repeated the selection process with the amino acids for helix 16. I moved back to the Protein > Primary tab and selected the amino acids that corresponded to Helix 1, and unchecked backbone. I moved the Atoms Size slider to 100. I selected the amino acids in helix 16 to apply the sizing change to them as well.

Figure 6: An image showing the proximity of helix 1 and helix 16 to the oxidized guanine base. Note that helix 16's atoms are in contact with the oxidized guanine, likely indicating that it is responsible for recognizing the damage.

Questions

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?
   • It is possible to differentiate between the DNA and protein components. The hOGG1 structure's proteins are displayed in the space-filling form, but the DNA is still visible in the ball and stick model (Fig. 1). Therefore, the DNA appears much smaller and thinner than the protein, making it easily distinguishable from the protein.
2. hOGG1 contains multiple sulfur atoms.
   1. Identify the name and sequence number of one of the amino acids in the structure that contains a sulfur atom.
      • The sulfur atom in figure 2 is part of cysteine at position 241.
   2. Is the sulfur atom located in the backbone or in the side chain of the amino acid?
      • The sulfur atom is located in the side chain of the amino acid (Fig. 3).
3. List the 13 amino acids numbered 105 through 117 in order.
   • See table 1.
4. Within a protein chain, amino acids form local structures called secondary structures.
   1. Are helices, sheets or coils present in hOGG1? Describe the color that represents the secondary structures you observe.
      • Helices, sheets, and coils are all present in hOGG1. Helices are colored pink, sheets are colored yellow-orange, and coils are colored a darker blue-purple color.
   2. Amino acids 105 through 117 fold into one of the secondary structures. Which secondary structure do they fold into?
      • Amino acids 105-107 fold into an alpha helix (Fig. 4).
5. 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.
   • The negatively charged amino acids are located on the outside (exposed) side of the protein (Fig. 5). This suggests that the cellular environment surrounding this protein is hydrophilic and made of a large amount of water. The amino acids on the outside will likely be exposed to the environment that is suitable to them. Negatively charged amino acids are hydrophilic, so the environment must be hydrophilic as well. If the environment was hydrophobic, the hydrophilic amino acids would repel the environment, and likely curve inwards so that they were not facing the outside.
6. Now let’s explore 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.
   1. How many DNA base pairs can you count within this double helix?
      • I can count 13 DNA base pairs within the double helix.
   2. How many DNA bases are unpaired (not paired to its partner on the other strand)?
      • There is one DNA base that is unpaired.
   3. 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 oxidized guanine is towards the center of the hOGG1 protein with the protein wrapping almost fully around it. It is also in the middle of the double helix. This suggest that hOGG1 uses the presence of oxidized guanine to determine whether or not a DNA base is damaged or not. The presence of it indicates the that base is damaged, and the absence of it indicates that the base is undamaged.
7. 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.
   • You are more likely to find the amino acids that recognize damaged guanine bases in Helix 16. Helix 16 is directly next to the oxidized guanine, and has a few atoms that are in direct contact with it (Fig. 6). Helix 1, on the other hand, is on the outside of the atom, quite far away from the oxidized guanine. The helix that is closest to the oxidized guanine will likely be the one responsible for identifying the damage, making it likely that helix 16 contains the amino acid that recognize the damaged guanine bases.
8. 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). We will compare these two structures to understand how hOGG1 can effectively distinguish between 8-oxoguanine and guanine.
   1. Which atom in the base portion of 8-oxoguanine is directly contacting glycine 42? Draw this interaction.
   2. What does the interaction between 8-oxoguanine and glycine 42 indicate about how hOGG1 differentiates between 8-oxoguanine and guanine?
   3. 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.

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Nicole Anguiano
BIOL 368, Fall 2014

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