Gene Mutations

Mutation Vocabulary Gene Mutation Chromosomal Mutation Point Mutation Substitution Silent Missense Nonsense Frameshift Insertion Deletion Duplication Deletion Translocation Inversion

Chromosomal Mutations

How do genes control metabolism In 1909, Archibald Garrod first proposed the relationship between genes and proteins. “Genes dictate phenotypes through enzymes that catalyze specific chemical processes in the cell.” Example: Alkaptonuria – Urine appears dark red because it contains alkapton, that darkens upon exposure to air. Wild Type Individuals have an enzyme to break the chemical down. Mutant Individuals do not produce the enzyme, thus unable to metabolize alkapton.

Beadle and Tatum One Gene – One Enzyme hypothesis : the Function of one gene is to dictate the production of a specific enzyme.

Transcription in Prokaryotes RNA polymerase cannot initiate transcription on its own. A protein (sigma) must bind before transcription can begin. RNA Polymerase + Sigma = HOLOENZYME When a holoenzyme + DNA mix, the enzyme will attach to only specific regions on the DNA which they now refer to as PROMOTERS. Specifically positions 10 and 35 nucleotides upstream from the gene. The sigma appeared to be responsible for guiding the RNA polymerase to specific locations. The sigma will release once initiation has commenced.

Promoter Sequence 20 – 25 base pairs long. Similar segment of DNA had a series of bases identical or similar to TATAAT. Referred to as the – 10 box.

Translation in Prokaryotes A large and small subunit assemble onto the mRNA. The small subunit will attach to what is called the Shine-Dalgarno Sequence on the mRNA. A 6 base sequence upstream 8 bases from the AUG start codon. The rRNA sequence within the small subunit will attach. Shine-Dalgarno Sequence – AGGAGG Anti Shine-Dalgarno Sequence (found on the ribosome) - UCCUCC

Translation in Prokaryotes The large subunit will attach and attract the first tRNA molecule. Loading in at the P-site of the ribosome.

Repairing Mistakes in DNA Synthesis Replication forks work at 50 bases per second Errors = one mistake per billion HUMAN REPLICATION 6 billion nucleotides Cells are replicated to create trillions of cells

DNA Polymerase Proofreading in Prokaryotes DNA polymerase acts as an exonuclease – (an enzyme that removes nucleotides from DNA) DNA polymerase III can remove nucleotides only from the 3’ end of the DNA, and only if they are not hydrogen bonded to a base on the complementary strand. If a wrong base is added during DNA synthesis, the enzyme pauses, removes the mismatched base that was just added, and then proceeds with synthesis.

Eukaryotic DNA polymerases Have the same type of proofreading ability – reduces error rate to about 1 in 10 million bases. At this rate there would be 600 mistakes every time a human cell replicated

Three Types of DNA Repair 1. Mismatch Repair 2. Thymine Dimer Repair 3. Excision Repair

Mismatch Repair When DNA polymerase doesn’t fix the problem, other enzymes spring into action. Responsible for “mismatch repair” The first repair enzyme is known as mutS. “mutatorS”

Which base is right? Hypothesis: At the conclusion of a replication process, a methyl group is added. So the proofreading enzyme will remove the nucleotide from the unmethylated strand.

Xeroderma Pigmentosum: A Case Study (DNA Repair Disorder) An autosomal recessive disease in humans. Extreme sensitivity to UV light. Skin will develop leasion after even slight exposure to sunlight. UV Light will cause a covalent bond to form between adjacent Thymines on a DNA strand. Creates a kink in the secondary structure of DNA. Causes a stall in the replication fork during replication.

The Study Cells of “normal” individuals versus cells of XP individuals. Exposed cells to UV radiation. Added radioactive Thymines to the cell which should be incorporated IF repair occurs. High amount of radioactive Thymines in the normal and virtual no radioactive thymines in the XP individuals.

DNA Excision Repair Uvr A, Uvr B, Uvr C, and Uvr D “Ultraviolet Light Repair”

Direct DNA repair DNA photolyase

DNA Mismatch Repair and Cancer (DNA Repair Disorder) Colon Cancer Variation = Hereditary nonpolyposis colorectal cancer (HNPCC) runs in families Tumors will develop on the colon, ovary, and other organs by the age of 50 1990 – researchers mapped the susceptibility to an area on chromosome 2

Do humans have mismatch repair genes? The research accelerated when mutS gene was identified and then research found a similar gene in a yeast genome. The genes were so similar, they called them homologous. Using the sequences from the genes, they located a similar sequence in the human gene – known as hMSH (human mutS homolog) Mapped to the same region on chromosome 2 as the HNPCC susceptibility gene.

Link between cancer and mismatch repair Cells from these patients have a mutation rate 100 times the normal. People who inherit a nonfunctional copy of the hMSH gene have a genetic predisposition for developing HNPCC. Evidence: Individuals who have this form of colon cancer have uneven repeats of sequences in their DNA (usually fixed in DNA repair).

Ataxia Telangiectasis (AT) Defect in the enzyme KINASE. Cells proceed through the checkpoints. (high mutation rate) Radiation Sensitivity Increased risk of breast cancer. Any problems?

Potential Benefits to this research If individuals with mutant forms of hMSH can be identified early in life, dietary changes and therapy could significantly reduce their risk of developing cancer

Semi-conservative Replication Meselson and Stahl Experiment