Essay Sample about BRCA1 Gene

Abstract

The tumor suppressor gene BRCA1 is a large protein that is implicated to play a significant role in stabilizing genomic instability. However, because of its size, disordered structure, and multiple sites for protein-DNA & protein-protein interactions, there is little information about the central region of BRCA1. Here, the focus was about the DNA binding patterns of BRCA1 DBD1 to investigate toxicity in the missense mutation, R496H. A Ni-NTA agarose column was used to purify DBD1 and ran it in an SDS-PAGE gel to investigate concentration, and then utilized EMSA to observe binding strength. Our results do not support toxicity in the missense mutation, R496H, because it was not expressed during mutagenesis.

Introduction

BRCA1 is a tumor suppressor gene that encodes for 1,863 amino acids that participate in DNA double strand break repair. BRCA1 is implicated in other functions such as transcription coupled repair, DNA damage signaling, and apoptosis. It is a largely unstructured protein with little information known of the central region except that there are DNA binding sites and protein binding sites. The two sites in the central region binds non-specifically to DNA, termed DNA binding domain (DBD). The first DNA binding site, DBD1, constitutes residues 330-554, and DBD2 constitutes residues 894-1057. The proteins BRCA1 binds to include BARD1 and Rad51.

BRCA1 mutations have been implicated in breast and ovarian cancers. BRCA1 is too large to purify by itself because it is difficult to isolate a high-quality protein sample. Because of the difficulty in whole protein purification, there is an abundance of unclassified mutations with unknown effects. So, we sought to experimentally assess the DNA binding patterns of BRCA1 DBD1 to determine if the missense mutation, R496H, is toxic.

Using Pymol, DBD1 was overlayed with the R496H variant, and the image illustrated clear structural differences between the wild-type DBD1 and mutated DBD1 because there is an amino acid change from arginine to histidine. Histidine has an imidazole ring while arginine does not. 

In the site-directed mutagenesis, the R496H mutation was not incorporated. The amino acid result of the R496H mutagenesis was PADQQTETQTPPDQ while the amino acid sequence of the wild-type DBD1 was EPQIIQERPLTNKLK. This means the mutagenesis failed because the arginine did not mutate into a histidine, the arginine instead mutated into a threonine.

After the column was finished, the NanoDrop was used to measure the protein concentration from the dialysis buffer, yielding an absorbance of 0.112. The calculated result was 0.673 μM. This was calculated using the molar extinction coefficient of 18,115 M-1 cm-1 and Beer’s law:

A/ε=C

Where A is absorbance, ε is the molar extinction coefficient, and C is concentration. Using ImageJ, the estimated purity of R496H protein in the elution lane was approximately 17% pure.

EMSA was utilized to observe the binding strength of wild type DBD1 and R496H DBD1. Wild-type (figure 3) and R496H (figure 5) DBD1 concentration goes from highest volume added (left) to lowest volume added (right). There are no noticeable super complexes.

Discussion

The mutation, R496H, was not incorporated. This may be due to an improper melting temperature of the primer, which was 6°C - 7°C lower than what the protocol desired, which was 78°C. Because the temperature was too high, this could have made the primers unable to bind to the template.

The purity of the elution of the R496H DBD1 protein was low compared to supernatant DBD1. This could be due to the Ni-NTA beads were shrinking during the experiment because of the beads drying out, which may have led to a lower concentration of DBD1 protein attaching to the beads. The Ni-NTA beads were not supposed to dry out at any point during the experiment since the size would decrease, therefore allowing less protein to bind to the beads. Because the band was so faint on the gel, it was assumed that the DBD1 protein was impure.

In EMSA, there was binding but the data was skewed due to a distortion in the gel, as well as the stain on the gel casting plate which all affectted lanes 1, 2, 3 and 9. A likely reason for the distortion in the gel was because there was not enough running buffer in the tank. From this, the conclusion was that DNA and the wild-type DBD1 do bind together.

The mutagenesis was unsuccessful, causing the entire experiment to assess and analyze a wild-type DBD1 instead of the mutated R496H DBD1. Hence, there remains limited information about whether the R496H DBD1 variant is toxic.

Experimental Procedures

3D Modeling

The AlphaFold Protein Structure Database was utilized to predict the structures of DBD1, DBD2 and the R496H DBD1 variant. Pymol was then applied to visualize all 3 structures as a cartoon. After, DBD1 was aligned with the R496H variant in a 600x600 resolution image.

Site-directed mutagenesis

The BRCA1 R496H variant primer was designed in the computer software Snapgene, which made the primer 45 bases long and had a melting temperature between 71°C to 72°C. The Agilent Quikchange II Site-Directed Mutagenesis kit was used to create the mutation, which had a 10x polymerase buffer, dNTP mix, F/R primers, plasmid DNA and a Pfu polymerase. After all the components were added and the protein was PCR amplified, 1 µL of a Dpn I restriction enzyme was added directly to the PCR product and was incubated at 37°C for an hour. Subsequently, the Dpn I digested DNA was digested into XL-1 blue competent cells, and then was transformed again into BL21 (DE3) cells for protein expression and purification in a Ni-NTA agarose column.

Protein expression and purification

NiCo21 (DE3) E. coli strain cells were purified in a column using Ni-NTA agarose beads. After the column was equilibrated using water and buffer A1 (20 mM NaPi, pH 7.5/ 300mM NaCl/ 30mM imidazole/ 1 mM DTT/ 5% glycerol/ 1% NP-40), 250 mL of the DE3 cells was resuspended in buffer A1 and added to the beads, which was incubated at 4°C for 30 minutes. The Ni-NTA beads were washed three times with buffer A1 and buffer A2 (20 mM NaPi, pH 7.5/ 30 mM NaCl/ 30 mM imidazole/ 1 mM DTT/ 5% glycerol) and then eluted with 2 mL of elution buffer (20 mM NaPi, pH 7.5/ 30 mM NaCl/ 250 mM imidazole/ 1 mM DTT/ 5% glycerol) and rested for 15 minutes at 4°C. The supernatant, first wash of buffer A1, first wash of buffer A2, elution buffer, and buffer B1, 30 µL each, were analyzed using 12% SDS-PAGE and 10 µL of 4x SDS loading dye.

EMSA

During EMSA, a 5% Native 1x TBE gel (30% acrylamide/ 10x TBE/ MQ water/ APS/ TEMED) was utilized. The gel ran between 80-100V at 4°C for approximately 40 minutes. Each sample included 10 µL of 1 µM Cy3-labeled DNA, 10 µL of 6x OrangeG, DBD1 protein and reaction buffer, which created a total volume of 60 µL. Each lane was loaded with 16 µL of sample. Both wild-type and R496H DBD1 proteins were used in separate experiments in varying volumes.

Analysis

Following EMSA and SDS-PAGE, ImageJ was used to analyze the gel pictures while, GraphPad Prism 9 was used to analyze the binding results taken from ImageJ. From GraphPad Prism 9, the DNA binding fraction vs DBD1 concentration graphs.