Gene Mutations

Mutation Vocabulary

Gene Mutation• Point Mutation

– Substitution– Silent– Missense– Nonsense

• Frameshift– Insertion– Deletion

Chromosomal Mutation• 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