1. 1

2. 1.Both prokaryotes and eukaryotes have enzyme-based DNA repair systems that prevent mutations and even death from DNA damage. 2.Repair systems are grouped by their repair mechanisms. Some directly correct, while others excise the damaged area and then repair the gap. Chapter 19 slide 2

3.  Three kinds: 1. Damage reversal 2. Damage removal 3. Damage tolerance/ Postreplication repair/Recombinational repair

4. Damage reversal

5.  Light-dependent repair or photoreactivation of DNA in bacteria is carried out by a light activated enzyme called DNA photolyase  light energy actives the enzyme to cleave the covalent cross-links  Photolyase will bind to thymine dimers in the dark, but it cannot catalyze cleavage of the bonds joining the thymine moieties  Photolyase also splits cytosine dimers and cytosine- thymine dimers

7. Damage by alkylation (usually methyl or ethyl groups) can be removed by specific DNA repair enzymes. a.For example, O 6 -methylguanine methyltransferase recognizes O 6 -methylguanine in DNA, and removes the methyl group. b.Demethylation restores the base to its original form.

9. Damage removal

10.  step 1, a DNA repair endonuclease or endonuclease- containing enzyme complex recognizes, binds to, and excises the damaged base or bases in DNA.  step 2, a DNA polymerase fills in the gap by using the undamaged complementary strand of DNA as template.  step 3,the enzyme DNA ligase seals the break left by DNA polymerase to complete the repair process

12.  There are two major types of excision repair: 1. Base excision repair systems remove abnormal or chemically modified bases from DNA, whereas 2. Nucleotide excision repair pathways remove larger defects like thymine dimers. 3. Other type : Mismatch repair  Both excision pathways are operative in the dark

13.  a- removal of the damaged base by a DNA glycosylase.(a group of eight enzymes,each one responsible for identifying and removing a specific kind of base damage.) b- removal of its sugar-phosphate in the backbone, by AP endonuclease and phosphodiesterase producing a gap: an AP site (apurinic/apyrimidinic).

14.  c- replacement with the correct nucleotide. Done by DNA polymerase using the other strand as a template. d- ligation of the break in the strand by ligase enzymes  Eg: presence of uracil in DNA

16.  30 distinct proteins that function as a large complex called the nucleotide excision repairosome (NER)  The most important DNA repair pathway  The sole repair system for bulky DNA lesions, which creates a block to DNA replication and transcription  Does not require light

17. a. In E. coli, NER corrects pyrimidine dimers and other damage-induced distortions of the DNA helix. b.The proteins required are UvrA, UvrB, UvrC and UvrD (encoded by genes of the same name) c.A complex of two UvrA and one UvrB proteins slides along the DNA. When it encounters a helix distortion, the UvrA subunits dissociate, and a UvrC binds the UvrB at the lesion. d.When UvrBC forms, the UvrC cuts 4–5 nucleotides from the lesion on the 3’ side, and eight nucleotides away on the 5’ side. Then UvrB is released and UvrD binds the 5’ cut end. e.UvrD is a helicase that unwinds the region between the cuts, releasing the short ssDNA, while DNA polymerase I fills the gap and DNA ligase seals the backbone. f.In yeast and mammalian systems, about 12 genes encode proteins involved in excision repair.

18. 台大農藝系 遺傳學 601 20000 Chapter 19 slide 18 Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

19.  Incorrect bases incorporated as a result of mistakes during DNA replication ( base mispairs, short insertions and deletions ) are excised as single nucleotides by a group of repair proteins which can scan DNA and look for incorrectly paired bases (or unpaired bases )  Since methylation occurs post-replication, repair proteins identify methylated strand as parent, remove mismatched bases on other strand and replace them

20.  Before DNA replication, the adenines in the sequence GATC are methylated (becoming N6- methyladenine). After replication, the new strand is not methylated, and therefore the molecule is known as hemimethylated DNA (because only one strand is methylated). The use of methylation is how E. coli identifies its old strands.

22.  before replication is completed, there is a distinction between the old and new strands. The way to tell them apart is that the old strand is methylated. To go on with this, out of the mismatched pair farther down the line, GT, the thymine would be the mistake that needs to be fixed.

24.  Here the single-stranded DNA is degraded by exonuclease after the uvrD unwinds the DNA between the nick and the mismatched base. The exonuclease removes part of the strand starting at GATC and continues all the way to the mismatch. This leaves a big gap in the strand. The uvrD is a helicase responsible for unwinding the two strands.

25.  Polymerase elongates the DNA, filling the gap, and goes right through the area where the mismatch was, correcting the lesion. Ligase then seals the nick at the end.

27. Damage tolerance/ Post replication repair/Recombinational repair

28.  In step 1, a DSB is introduced into one of the DNA substrates.  In step 2, the DSB is processed by either a helicase, a nuclease, or a combination of both, to yield a free 3’-ssDNA overhang(s)  In step 3, this free 3’-terminal end is homologously paired with its homologue by a RecA-like protein to form an intermediate known as a joint molecule  Following joint molecule formation, pairing of the displaced complementary strands results in the formation of characteristic intermediates junctions (step 4)  In step 5, thejunction(s) is extended unidirectionally by branch migration proteins.  Finally, in step 6, the junction is cleaved by a resolvase to yield recombinant products

31. The SOS response in bacteria results when specific base pairing cannot occur. It allows the cell to survive otherwise lethal events, but usually at the cost of incurring new mutations. E. coli SOS is controlled by two genes, lexA and recA. (Mutants in either of these genes have their SOS response permanently turned on.) i. When no DNA damage is present, LexA represses transcription of about 17 genes with products involved in various types of DNA repair. ii. Sufficient DNA damage activates the RecA protein, which stimulates LexA to autocleave, removing repression of the DNA repair genes. iii. After damage is repaired, RecA is inactivated, and newly synthesized LexA again represses the DNA repair genes.