Page 1 of 10 DNA Damage and Repair I Jason Kahn, Biochemistry 465, 4/18/06 Outline: • General considerations about DNA damage responses • Chemistry: Sources, types, and consequences of DNA damage • Repair Mechanisms: Direct reversal, BER, NER (next time: Mismatch repair, recombination-mediated repair, coupling to transcription) General considerations DNA damage is an inevitable consequence of using water in the cytoplasm, breathing air, eating food, and being exposed to sunlight and cosmic rays. Cells have active mechanisms to repair the damage, and defective repair mechanisms can lead to profound developmental problems as well as greatly increased cancer susceptibility. Examples of human diseases include HNPCC (human non-polyposis colon cancer, due to a defect in mismatch repair), XP (Xeroderma pigmentosa, due to defective nucleotide excision repair, NER). We've already seen some of the responses to damage when we studied checkpoints in the eukaryotic cell cycle. Here's a flowchart for the responses:Page 2 of 10 "Error free" repair uses the other strand of the DNA or another copy available in the cell to make a perfect new version with the same information as existed before the damage occurred. "Error-prone repair" can be trans-lesion synthesis, random joining of DNA ends: anything that doesn't use the original information in the DNA to make a clean copy. As we've said, DNA damage doesn't really matter until the cell actually tries to use the information for replication or transcription. We won't deal with it further. Balance between apoptosis and allowing some level of mutation is quite tricky… Chemistry of DNA Damage Sources/types of DNA damage: • Adducts like 8-oxo-guanosine (from ROS), thymine dimers (from UV), psoralen crosslinks (from picking vegetables in the sun; picture on web page), or benz-pyrene diol epoxide adducts to G (from barbecuing, via epoxidation in the liver. Thank you, liver). Cis-syn T<>T BPDE from http://www.nyu.edu/its/pubs/connect/archives/98spring/broydedna.html 8-oxoG miscoding from Brieba et al., 2004. EMBO J.23:3452-61 (Ellenberger group) • Mismatches and small loops, from the occasional C to U deamination reaction, misincorporation or slippage error (repeated synthesis of one or a few template nucleotides, see below) in DNA polymerization. • Strand breaks, with double-strand breaks (DSB's) being much worse, from ionizing radiation or collapsed replication forks.Page 3 of 10 Consequences of DNA damage: • Translesion synthesis or copying of mismatches/loops leads to substitution or frameshift mutations. (And it’s not until the DNA is copied that this kind of damage really causes a problem.) For example, triplet repeat expansion = repeated loop slippage is the root of many neurological diseases, including Fragile X syndrome and Huntington's disease. The diseases can be caused by misfolding of the resulting protein or because of the properties of the DNA/RNA. Interesting example of a genetic disease in which symptoms get worse with each generation as the repeat expands; initially clinicians pointed this out and basic scientists didn't believe it. Image from Wells et al., Nucleic Acids Research 2005 33(12):3785-3798; doi:10.1093/nar/gki697, no need to draw out. • Inappropriate end joining due to DSBs can give chromosomal translocations: for example, chronic myelogenous leukemia is caused by a translocation that produces the BCR-ABL fusion protein, a tyrosine kinase that leads to impaired control of the cell cycle. Treated with the kinase inhibitor Gleevec. [No need to redraw picture, which is from users.rcn.com/jkimball.ma.ultranet/ BiologyPages/C/CML.html] • Excessive DNA damage leads to apoptosis, or plain old death if the DNA is too heavily sheared. Some bacteria are highly resistant because they have multiple copies of their genomes and can reassemble them after they are cut up.Page 4 of 10 Review general ideas and lead in to repair: • DNA damage is inevitable. • Miscoding lesions and other types of damage can kill or dys-regulate the cell. (Many modifications, like 6-methylA, are normal, not miscoding). • The consequences of a type of lesion depend on three things, something like: Consequences = (rate of appearance – rate of repair) ×"seriousness" • As might be expected, there are diverse repair mechanisms to handle the diverse types of damage. • But…as in most of metabolism, a large number of metabolites feed in to a small number of common intermediates, so the number of pathways is much smaller than the number of adducts. Mechanisms of Repair Roughly in order of how complicated they are, and in order of decreasing specialization. Common types of damage have dedicated pathways, less common types have more general pathways that may not be as fast or accurate. 1. Direct Reversal. 2. Base excision repair = BER 3. Nucleotide excision repair = NER or short patch repair 4. Mismatch repair, in E. coli it's methyl-directed mismatch repair, MMR 5. Recombination-mediated repair of crosslinks, DSB's 1. Direct Reversal: Fix the damaged base/adduct in situ Specialized handling of commonly-encountered simple adducts. This is the only pathway that does not require DNA polymerase activity. Example: Ada methyltransferase The Ada methyltransferase repairs O6-methylG (miscoding lesion…students should draw it and figure out why, again) and phosphotriesters. It has a nucleophilic active-site Cys that ends up methylated, and the DNA is regenerated.Page 5 of 10 Note that the protein methylation is irreversible: strictly, this is a stoichiometric reaction, not an enzymatic one. This illustrates a general principle of repair: it's expensive, and evolution has decided that it's worth it! The Ada-Cys-Me is put to use, however – it's a transcription factor that activates it's own expression and other repair pathways! Example: DNA photolyase This enzyme repairs thymine photodimers in all three kingdoms. We don’t have it, sigh. In some cases, photolyase can block or target repair of adducts even if it is inactive. (One of?) the earliest repair mechanisms discovered (1935), even though at the time they didn't know about DNA: Why poor media? Slow growth avoids attempts to copy the lesions, allows time for repair. Mechanism of photolyase? Reduced FADH- absorbs blue light photon [actually it is usually excited by an "antenna chromophore," a bound MTHF = methenyl tetrahydrofolate, viz.
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