BC34C DNA Repair Mechanisms Dr Mark Ashby Room 45/lab2. [email protected][email protected] Lecture 1 Mon Oct 27 8am Lecture 2 Tue Oct 28 10am Lecture 3 Mon Nov 5 10am Slide 2 References. wwwbiochem.uwimona.edu.jm:1104/bc34c Genes VII Lodish et al. Available in the library and copies in library and Dr Royes book loan scheme. Molecular Cell Biology (HR). 4 th Edition by Lodish et al. WH Freeman and co. should be 3 copies in the library (soon?). Available online at www.ncbi.nlm.nih.gov/entrez/. www.ncbi.nlm.nih.gov/entrez/ An Introduction to Genetic Analysis 7the edition, Griffiths et al. (library at some time). Also available online. Molecular Biology of the Cell, Fourth Edition by Bruce Alberts et al. (Arriving in the library at some time). Also available online. Slide 3 Slide 4 Q. What is it about the structure of DNA that enables damage to be repaired? Think about DNA being a double helix. If one or nucleotides in one strand are damaged or misincorporated then the other strand has the complementary sequence to correct the problems. Slide 5 Very basically repair systems must: A Recognise the problem B Remove the problem C Repair the problem Slide 6 Causes of mismatched bases Slide 7 Causes of Structural distortion in the double helix Slide 8 Repair mechanisms can often recognise structural distortions of DNA structure. These can be divided into several types. It is harder to recognise mismatches. Proofreading Direct repair. Very rare. Excision repair Mismatch repair Tolerance systems Retrieval systems Slide 9 Proofreading. This is an intrinsic property of almost all DNA polymerases and was dealt with in Mol Biol I. For example the proofreading activity of DNA pol III reduces the number of errors during replication by 1000 fold. Slide 10 Direct Repair of a UV-induced pyrimidine photodimer by a photolyase. The enzyme recognizes the photodimer and binds to it. When light is present, the photolyase uses its energy to split the dimer into the original monomers. Slide 11 Excision Repair. This is initiated by a recognition enzyme. They can either recognise damaged nucleotides or structural distortions. There are a number of different enzymes, some recognising general damage while others recognise specific types of damage (glycosylases and AP endonucleases). There are multiple excision-repair systems which deal with most of the damage. Slide 12 General properties of Excision repair Slide 13 Incision, the damaged structure is recognised by an endonuclease that cleaves the DNA either side of the damage. Excision a 5 3 exonuclease removes the damaged strand. Synthesis the ssDNA serves as s template for a DNA polymerase (polI) to make a replacement strand. DNA ligase seals the nick. Slide 14 Base Excision repair. Battery of enzymes called DNA glycolases each recognising a specific type of altered base, catalysing its hydrolytic removal. At least 6 different glycolases, including those that remove deaminated Cs, deaminated As, akylated or oxidised bases, bases with open rings, where C double has been converted to C single bond. Slide 15 Comparison of two major DNA repair pathways. On the left Base Excision repair. Slide 16 The most common lesion is depurination which leaves the deoxyribose sugar without a base. AP endonuclease and phosphodiesterase work directly on this. The loss of G or A which has resulted from the cleavage of the glycosidic bond between the deoxyribose and the base, releasing the base. Reapir starts at the 3 rd stage of the base excision pathway. Slide 17 Nucleotide excision repair. This is used to repair bulky lesions. This can include UV induced pyrimidine dimers like T-T, T-C, C-C and bases that have reacted with organic carcinogens. A large multienzyme complex scans for a distortion rather than for a specific base, so it can recognise a number of different lesions. The phosphodiester bond is cleaved either side of the distortion, a helicase peels away the offending strand. It is then repaired by pol I and ligase. Slide 18 An example nucleotide excision repair is the Uvr system in E.coli. This involves the products of three genes, uvrA, B, C. They code for components of a repair endonuclease. Slide 19 Slide 20 UvrA 2 B recognises pyrimidine dimers and other bulky lesions. It probably translocates along the DNA (needing ATP) until it encounters a distortion. An ATP dependant conformational change occurs in the damaged region, producing a kink in the DNA backbone. UvrA dissociates (needing ATP) and UvrC (has endonuclease activity) joins UvrB. The interaction is thought to open up a space within the DNA for the catalytic site of the enzyme to get to the target. Slide 21 Model of complex formed between bacteriophage T4 endonuclease V and a 13-bp DNA fragment containing a thymine-thymine dimer based on x-ray crystallographic analysis. UvrC is thought to bind in a similar fashion. Slide 22 The position of the cleavage is determined by the nature of the DNA damage. With thymine dimers UvrC cleaves two phosphodiester bonds. One 7-8 nucleotides from the 5 side and 3-5 from the 3 side. This also requires ATP. UvrD is a helicase that unwinds the DNA between the two cuts. It is probably DNA pol I that that excises the damaged strand. Repair is made by pol I and ligase. Slide 23 The average length of excised DNA is 12, it is called short-patch repair. Short patch accounts for 99% of excision repair events. The other 1% is long patch repairs. The excised strand can be 1500-9,000 nucleotides. Short patch is a normal function of the cell whereas long patch is induced by damage. Probably acting on lesions found near replication forks. (Can be associated with the SOS response. Some proteins, like umuDC induced by damage can allow error prone replication to take place). They allow error prone replication to take place (involving pol III).