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#45 Biochemistry DNA Repair / Transcription I Lecture for Kevin Ahern's BB 451/551





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Uploaded on Feb 28, 2012

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DNA Replication/Repair/Recombination (continued)
1. Repair of DNA damage is critical for cellular and organismal health.

2. One DNA error linked to human disease is the triplet repeat extension problem that is associated with Huntington's disease. In this syndrome, repeats of a three base sequence in the coding region of a protein "slide" during replication, causing variability in the numbers of the repeat and, consequently, variability in the protein(s) made from it.

3. Other error-related systems associated with cancer include HNPCC (colon cancer) and BRC-A (not mentioned in class), which is involved in DNA repair.

4. An important proto-oncogene that plays a role in ensuring DNA fidelity is p53. p53 is the guardian of the genome acts to stop the cell cycle if DNA replication has not completed properly. Then, acting as a transcription factor, it activates transcription/translation of DNA repair genes. If these genes can't fix the damage, p53 induces apoptosis.

5. The Ames test is used to test the tendency of a compound to cause mutation. It works by using a plasmid with a single base mismatch in a gene marker that stops expression to give the marker (blue color, for example). When cells carry this gene and then half are exposed to the test compound and have are not exposed. By comparing the rates of marker production, the tendency of a compound to cause mutation can be determined.

6. Recombination of DNA results in mixing and matching of DNA sequences. The process occurs most often between homologous sequences on different chromosomes. The process can be quite active during meiosis.

7. Recombination proceeds through formation of a Holliday junction. Holliday junctions form as a result of alignment of homologous sequences, followed by cleavage of strands on each chromosome, invasion of the strands into the opposite chromosome, movement of the junction, another cleavage reaction, followed by reformation of phosphodiester bonds.

8. Enzymes involved in recombination are called recombinases and are similar in function to the integrase of HIV.

9. Recombination is important for mixing of DNA sequences and processes, such as antibody diversity and for integration of retroviruses into host chromosomes.

Transcription Highlights
1. Transcription is the process where RNA is made using DNA as a template. Students should ABSOLUTELY not mix up or misuse the terms DNA Replication, Transcription, and Translation.

2. RNA polymerization requires an enzyme called RNA polymerase. It can start a chain without a primer, incorporates nucleotides into a growing chain in the 5' to 3' direction using phosphodiester bonds, and uses ATP, GTP, CTP, and UTP as starting compounds. The product of RNA polymerization is called a transcript.

3. The DNA strand that is complementary to the RNA made in transcription is called the template strand. The strand that has the same sequence (except T vs. U) is called the coding strand.

4. Transcription is controlled in cells and starts near sequences called promoters.

5. Promoters in E. coli (RNA polymerase binding sites adjacent to genes) function with widely varying rates of efficiency - some stimulating initiation of transcription every few seconds, others only one or twice per life cycle. One way for a promoter to control such events is via variation in conserved sequences. E. coli genes have two conserved sequences - one at -10 relative to the transcription start site (TATAAT) and another at -35 relative to the transcription start site (TTGACA).

6. The more closely a given promoter's sequence matches the consensus sequence of the -10 sequence, the more active the promoter is at initiating transcription. RNA Polymerase slides along the DNA and when the sigma subunit identifies a promoter site, it stops.

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