BMB 251 1st Edition Lecture 16 Outline of Last Lecture I. ClickersII. DNA replication originsIII. Replication unitsIV. Thymidine analog bromodeoxyuridine (BrdU)V. Origin Recognition ComplexVI. End Replication problemsVII. Replicative cell senescenceOutline of Current Lecture VIII. ClickersIX. DNA repairX. DepurinationXI. DeaminationXII. Base-excision repairXIII. Nucleotide-excision repairXIV. Homologous recombinationCurrent Lecture- Clicker Question 1: Comparing the following, which is true?o Replication is initiated at an origin by binding (prokaryotes) and phosphorylation of a subunit (eukaryotes) of the replication complex Both prokaryotes and eukaryotes proceed from the origin in both directions In each cell cycle, each of the origin of replication “fires” once- Clicker Question 2: Which bonds are broken during depurination/ deamination?o N-glycosidic- Clicker Question 3: Pyrimidine dimers do not disrupt base pairing. But they are extremely harmful to cells. Why?o They lead to very high rates of replication errors ANDo They inhibit replication by impeding DNA polymerase- DNA repair: set of processes that immediately correct changes in DNA o Importance is shown by several percent of coding genes is devoted to DNA repair functions and number of mutations increases when DNA repair gene is inactivated- Depurination: spontaneous reaction where purine bases’ (A and G) N-glycosyl linkages to deoxyribose hydrolyze  lose around 5000 purine bases per day- Deamination: spontaneous reaction in which cytosine is switched with uracil in DNA at a rate of about 100 bases per cell per dayThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- DNA bases also damaged by: encounters with reactive metabolite produced in cell, exposure to chemicals in environments and UV radiation (covalent linkage between two adjacent pyrimidine bases form)o If left uncorrected, these changes can lead to deletion of one or more base pairs or base-pair substitution in daughter DNA chain- When one strand of DNA is damaged, the complementary strand retains an intact cope of the same genetic info which is then used to restore correct nucleotides to damaged strand- Base-excision repair: involve DNA glycosylate enzymes, each of which can recognize a specific type of altered base in DNA and catalyze its hydrolytic removalo At least 6 types of these enzymes o “Flip out” nucleotides from helix, via smaller enzymes, which allows DNA glycosylase to probe bases for damage; these smaller enzymes are thought to travel along DNA and evaluate the status of each base  once a damaged base is found, it is removedo “Missing tooth” created by DNA glycosylase is recognized by AP endonuclease, which then cuts out two phosphodiester bonds in order to tak out a chunk of the backbone (creates a “nick”)o Complementary DNA strand is used to repair the gap left, sealed up by DNA ligase- Nucleotide excision repair: repairs damage caused by almost any large covalent reaction of DNA bases with large hydrocarbons; example: pyrimidine dimers caused by sunlight (H-bonds are distorted without being disrupted)o Large multienzyme complex scans DNA for distortion in double helix, not specific base changeso Once damage is found, it cleaves phosphodiester backbone of abnormal strands on bothsides of distortion an DNA helicase peels away single-strand oligonucleotide containing the lesion  large gap repaired by DNA polymerase and ligase- Direct chemical reversal of DNA damage can also be used  rapid removal of certain highly mutagenic or cytotoxic lesions- Cells have a way of directing DNA repair to DNA sequences most urgently needed o Link RNA polymerase to repair DNA damageo **Transcription-coupled repair fixes cell’s most important DNA sequences immediately - Cytosine can be methylated into uracil; 5-methyl cytosine can be methylated into thymine  causes mutations- When cell’s DNA is heavily damaged, cell employs emergency back-up polymerases to replicate through DNA damage o These are versatile but less accurateo Lack of exonucleolytic proofreading activity and less discrimination in choosing new nucleotides leads to less accuracy- Dangerous DNA damage occurs when both strands of double helix break, which would cause chromosomes to break down and genes lost during cell division if left untreatedo Nonhomologous end-joining: broken ends are brought together and rejoined via DNA ligation (generally loses one or more nucleotides at site of joining)o Although mutation occurs, it is deemed and “okay” process because so little of the genome codes for proteinsHomologous recombination: DNA is repaired using sister chromatid as template, occurring in newly replicated DNA only- DNA repair mechanisms are maximized in effectiveness by delaying progression of cell cycle untilDNA repair is complete  use of checkpoints (one step must be completed before next can begin)- DNA damage  increased synthesis of some DNA repair