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Dynamic Probes of Physical States in Live Cells Presented by Dr. Brett Helms, Molecular Foundry

Dynamic Probes of Physical States in Live Cells Presented by Dr. Brett Helms, Molecular Foundry Lawrence Berkeley National Laboratories Berkeley, California Part of AMSEC ’ s MAD Seminar Series M aterials S cience S eminar Monday, April 11th

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Dynamic Probes of Physical States in Live Cells Presented by Dr. Brett Helms, Molecular Foundry

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  1. Dynamic Probes of Physical States in Live Cells Presented by Dr. Brett Helms, Molecular Foundry Lawrence Berkeley National Laboratories Berkeley, California Part of AMSEC’s MAD Seminar Series Materials Science Seminar Monday, April 11th 5:15pm in CF110 / Refreshments at 5:00pm in CF110

  2. Replication of Genetic Code • Strand separation occurs first • Each strand serves as a template • for the synthesis of a new strand • Synthesis is catalyzed by enzymes • known as DNA polymerases • Newly made DNA molecule has one • daughter strand and one parent strand. “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material” Watson and Crick, in their Nature paper,1953

  3. How would YOU go about determining the mechanism of DNA replication????? What would a geneticist do? What would a biochemist do?

  4. Figure 5-31 Action of DNA polymerases. Page 99

  5. Figure 5-32a Replication of duplex DNA in E. coli. Page 100

  6. Figure 5-32b Replication of duplex DNA in E. coli. Page 100

  7. Here’s a computer modelhttp://www.youtube.com/watch?v=4jtmOZaIvS0 Overview of DNA and replication http://207.207.4.198/pub/flash/24/menu.swf This is a pretty good outline: http://www.youtube.com/watch?v=teV62zrm2P0&NR=1 Another one with review questions http://www.wiley.com/college/pratt/0471393878/student/animations/dna_replication/index.html

  8. Molecular Mechanisms of Spontaneous Mutagenesis • Deamination • Very slow reactions • Large number of residues • The net effect is significant: 100 C  U • events /day in a mammalian cell • Depurination • N-glycosidic bond is hydrolyzed • Significant for purines: 10,000 purines • lost/day in a mammalian cell • Cells have mechanisms to correct most of these modifications.

  9. Some well-characterized nonenzymatic reactions of nucleotides.

  10. Some well-characterized nonenzymatic reactions of nucleotides.

  11. Molecular Mechanisms of Oxidative and Chemical Mutagenesis • Oxidative damage • Hydroxylation of guanine • Mitochondrial DNA is most susceptible • Chemical alkylation • Methylation of guanine • Cells have mechanisms to correct most of these modifications

  12. Molecular Mechanisms of Radiation-Induced Mutagenesis • UV light induces dimerization of pyrimidines, this may be the main mechanism for skin cancers • Ionizing radiation (X-rays and -rays) causes ring opening and strand breaking. These are difficult to fix • Cells can repair some of these modifications, but others cause mutations. Accumulation of mutations is linked to aging and carcinogenesis

  13. Formation of pyrimidine dimers induced by UV light. (b) Formation of a cyclobutane pyrimidine dimer introduces a bend or kink into the DNA

  14. Review of DNA Repair Mechanisms http://www.nature.com/nature/journal/v421/n6921/full/nature01408.html Article to read for discussion on Friday: http://www.nature.com/nature/journal/v468/n7322/full/nature09428.html#/base- excision-by-solvent-exposure Cute animations Nucleotide Excision Repair

  15. DNA damage (black triangle) results in either repair or tolerance. • a, During damage tolerance, damaged sites are recognized by the replication machinery before they can be repaired, resulting in an arrest that can be relieved by replicative bypass (translesion DNA synthesis). • b, DNA repair involves the excision of bases and DNA synthesis (red wavy lines), which requires double-stranded DNA. Mispaired bases, usually generated by mistakes during DNA replication, are excised as single nucleotides during mismatch repair. A damaged base is excised as a single free base (base excision repair) or as an oligonucleotide fragment (nucleotide excision repair). Such fragments are generated by incisions flanking either side of the damaged base. Nucleotide excision repair can also transpire in some organisms by a distinct biochemical mechanism involving only a single incision next to a site of damage (unimodal incision). • c, The cell has a network of complex signalling pathways that arrest the cell cycle and may ultimately lead to programmed cell death.

  16. Diseases • colon cancer • cellular ultraviolet sensitivity • Werner syndrome (premature aging, retarded growth) • Bloom syndrome (sunlight hypersensitivity)

  17. Damage of the double helix • Single strand damage • information is still backed up in the other strand • Double strand damage • no backup • can cause the chromosome to break up

  18. Page 1173 Figure 30-51 Types and sites of chemical damage to which DNA is normally susceptible in vivo. Red, oxidation; blue, hydrolysis; green, methylation.

  19. Endogenous and exogenous alkylating agents (tobacco smoke, some anticancer drugs): O6-alkylguanine has a different pattern of H-bond donor and acceptor atoms than the parent guanine base. As a result, it basepairs with T instead of C, giving rise to G  A transition after the second round of replication:

  20. Single strand repair • Nucleotide excision repair • a large multienzyme compound scans the DNA strand for anomalities • upon detection a nuclease cuts the strand on both sides of the damage • DNA helicase removes the oligonucleotide • the gap is repaired by DNA polymerase and DNA ligase enzymes

  21. O6-alkylguanine DNA alkyltransferase (AGT) Directly repaires alkylation damage (O6-alkylguanines) by transferring the O6-alkyl group from damaged guanine in DNA to a Cys residue in the AGT active site in a stoichiometric reaction. The protein is inactivated via alkylation and undergoes proteolytic degradation.  AGT protein is highly conserved:  helix-turn-helix DNA binding motif  the alkylated base is “flipped” out of the helix to enter the hydrophobic alkyl-binding pocket of the protein  high metabolic cost for the cell is outweighed by the need to maintain genetic integrity

  22. Figure 30-52 The cyclobutylthymine dimer that forms on UV irradiation of two adjacent thymine residues on a DNA strand. Photolyase: repairs cyclobutane pyrimidine dimers. Uses the energy of light to catalyze the reversal of the cyclobutane bonds, producing intact DNA. Not very important in mammals.

  23. Figure 30-55 The mechanism of nucleotide excision repair (NER) of pyrimidine photodimers. Page 1176

  24. Single strand repair • Base excision repair • A base-specific DNA glycosylase detects an altered base and removes it • AP endonuclease and phosphodiesterase remove sugar phosphate • DNA Polymerase fills and DNA ligase seals the nick

  25. Figure 30-56 Action of DNA glycosylases. These enzymes hydrolyze the glycosidic bond of their corresponding altered base (red) to yield an AP site. Page 1177

  26. Figure 30-57 X-Ray structure of human uracil–DNA glycosylase (UDG) in complex with a 10-bp DNA containing a U·G base pair. Page 1178

  27. Base Excision Repair Used for repair of small damaged bases in DNA (AP sites, oxidized, deaminated, and methylated bases) Several steps are involved: a) modified base is excised by N-glycosylase to give an abasic site b) the abasic site is cleaved c) the resulting single-nucleotide gap is filled by DNA Polymerase d) DNA Ligase seals the nicks Example: uracil DNA glycosylase

  28. Figure 30-58The mechanism of mismatch repair in E. coli. Page 1179

  29. Mismatch Repair Mismatch Repair deals with correcting mismatches of normal bases. Steps in MMR: •Recognition of a mismatch •Identification of newly synthesized strand •Removal of mismatch•Gap repair by DNA Pol

  30. Figure 30-59 Regulation of the SOS response in E. coli. Page 1180

  31. Double strand repair • Nonhomologous end-joining • only in emergency situations • two broken ends of DNA are joined together • a couple of nucleotides are cut from both of the strands • ligase joins the strands together

  32. Double strand repair • Homologous end-joining • damaged site is copied from the other chromosome by special recombination proteins

  33. DNA repair enzymes • a lot of DNA damage -> elevated levels of repair enzymes • extreme change in cell's environment (heat, UV, radiation) activates genes that code DNA repair enzymes • For an example, heat-shock proteins are produced in heat-shock response when being subjected to high temperatures.

  34. Cell Cycle and DNA repair • Cell cycle is delayed if there is a lot of DNA damage. • Repairing DNA as well as signals sent by damaged DNA delays progression of cell cycle. ->ensures that DNA damages are repaired before the cell divides

  35. Chapter 8: Summary In this chapter, we learned about: • Function of nucleotides and nucleic acids • Names and structures of common nucleotides • Structural basis of DNA function • Reversible denaturation of nucleic acids • Chemical basis of mutagenesis

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