Bio 211 Exam 4: DNA damage and repair – Flashcards

-presence of deoxyribose rather than ribose in DNA makes it less susceptible to hydrolysis -damage on one strand can be corrected by the intact information on the other strand

-endogenous agents: chemicals formed within cells, often by metabolic processes - toxic byproducts -exogenous agents: chemicals found in the environment -DNA damaging agents that cause mutations are called mutagens which can lead to cancer by inactivating tumor suppressors and activating proto-oncogenes (and so may be called carcinogens)

-mutations can cause cancer and other diseases, or lead to cell death when transcription/translation is severely damaged -DNA repair mechanisms are found in all forms of life -cells invest a lot of energy in repairing DNA damage (a single double-stranded break (DSB) could require over 10,000 molecules of ATP to repair)

there are three types: UV-C (shortest), UV-B (intermediate), and UV-A (longest)

UV-C is particularly damaging, because it includes 260 nm, the wavelength of maximum absorption for DNA

causes most of the DNA damage in skin, though it accounts for only ~10% of the UV radiation reaching the earth's surface

-penetrates more deeply than UV-B, but not as effective as causing DNA damage -causes tanning -increased exposure = skin aging, wrinkling, increased risk of skin cancer

-two types of pyrimidine dimers (linkage of two pyrimidines in DNA) make up nearly all UV-induced DNA damage -cyclobutane pyrimidine dimer makes up ~75% of UV-induced damage -made up of two bonds between the C-5 and C-6 atoms of adjacent pyrimidines -"clog up" active site of RNA/DNA polymerases -major cause of melanoma - uncontrolled cell growth

-named because bond created between C-6 atom of the 3' pyrimidine and the C-4 atom of the 5' pyrimidine -causes major distortions in B-DNA structure, leading to transcription and replication problems -bends/drags the 3' nucleotide and causes major problems

gamma and x-rays cause many types of damage -cause double stranded breaks (DSB) -marked by phosphorylated H2AX -can lead to chromosomal rearrangements due to chromosome segments breaking and reattaching at inappropriate places ... high mutagenic dependability -majority of ionizing radiation damage is due to production of reactive oxygen species (ROS), which are highly reactive and damage biomolecules they encounter

many sites are vulnerable to damage of DNA: phosphodiester bonds, N-glycosylic bonds, exocyclic amine groups, sites attacked by ROSs, electron rich atoms that can be attacked by alkylating agents

cleavage of an N-glycosyl bond, which links deoxyribose to a base, leads to the formation of abasic sites (also called AP sites) (sites lacking a base) -~10,000 purine and ~500 pyrimidine bases are lost from DNA every day

apurinic/apyrimidinic sites

-conversion of bases to different bases -hydrolytic cleavage of bonds to exocyclic amines removes these amines from bases (deamination) -hydrolytic deamination of cytosine is estimated to take place 100-500 ties a day in mammalian cells -guanine and adenine deaminations together are thought to take place at a rate of 1-2% of those of cytosine -deamination of guanine to xanthine is a problem ... Xanthine cannot stably base pair with cytosine or thymine and so can cause mutations or arrest of DNA synthesis -deamination of adenine to hypoxanthine will convert a T-A to a C-G base pair (transition mutation) -deamination of cytosine to uracil will convert a C-G to a T-A base pair (transition mutation) -nitrous acid, formed from nitrites used as preservatives in processed meats, enchances deamination

-hydroxyl radicals can cause >80 kinds of oxidative base damages -8-oxoguanine and thymine glycol -10,000-11,500 oxidative damage events/day are estimated to happen in human cells

-can pair with cytosine or adenine -an 8-oxoG-C pair is fine, as it's still a G-C base pair, but an uncorrected 8-oxoG-A pair will be replicated to form a T-A base pair, resulting in a transversion mutation

-chemicals called alkylating agents transfer methyl, ethyl, or larger alkyl groups to electron-rich atoms in DNA -the product formed by attaching a chemical group to DNA is called an adduct - which changes the structure significantly

-Percival Potts noted many male patients with scrotal cancer had worked as chimney sweeps as children, leading him to conclude that there was a causal relationship between soot exposure and cancer -we now know soot contains polycyclic aromatic hydrocarbons (PAHs)

-some alkylating agents can form cross-links within one strand of DNA (intrastrand crosslinks) or corsslinks between the two strands of DNA (interstrand crosslinks) -nitrogen mustard gas is an example, as the crosslink is formed by the covalent linkage of guanines on opposite strands of DNA

-cisplatin, an interstrand cross-linker used as a chemotherapeutic agent -it inhibits replication and transcription -psoralen is used to treat skin cancers

-cross-linking agents, like cisplatin, are very effective at killing cancer cells (they are cytotoxic), but they are non-selective, meaning they kill cancer and healthy cells equally well -the hope of using them is that cancer cells are growing and dividing more vigorously than healthy cells, so the healthy cells will win out

-pharmaceutical, food, textile, petroleum, and other industries synthesize tens of thousands of chemicals every year for various applications -one of the most important things is to know if these promising compounds cause mutations

-determines if a chemical compound is mutagenic -A salmonella strain requiring histidine is incubated with rat liver extract to stimulate metabolism -the bacteria are plated on medium with a small amount of histidine with or without the chemical of interest to allow a few cell divisions to take place -the plates are scored for colonies. the presence of many colonies indicates that the mutant his gene reverted to wild-type due to increased mutation frequency caused by the potential mutagen -number of colonies correlated with mutations

fix specific legion or start from scratch

an enzyme called photolyase uses energy from blue light wavelength to disrupt the cyclobutane ring

-another photolyase repairs (6-4) photoproducts - not universal because there has not been a photolyase found in mammals and bacteria -instead, the whole damaged nucleotide is cut out using nucleotide excision repair

-repair requires the coordinated action of several enzymes to reverse DNA damage -repair of deaminated, oxidized, and alkylated bases requires one such pathway, called base excision repair (BER) -two classes: N-glycosylaes that cleave the N-glycosyl bond between the base and sugar

only cut the bond between a damaged base and its sugar at the AP site monofunctional enzyme

cut both the bond between a damaged base and its sugar and the bond between the sugar and the phosphate 3' to the site of damage bifunctional enzyme

-DNA glycosylase excises a base, producing an abasic site -AP endonuclease cleaves the DNA backbone, generating a free 3' OH and 5' deoxyribose phosphate -from here there are two potential mechanisms of repairing damage to fill the gap

-DNA polymerase delta or epsilon cooperates with the clamp loader and PCNA to add 2 or more nucleotides from the free 3' OH -flap endonuclease replaces the "flap" of DNA containing the abasic site -DNA ligase seals the DNA

-DNA polymerase beta (a polymerase specialized for DNA repair) adds one nucleotide to the free 3' OH and removes the 5' deoxyribose phosphate -DNA ligase seals the DNA

-NER is a pathway that involves the coordinated activity of several enzymes that work to remove and replace damaged nucleotides -removes bulky adducts from DNA --> pyrimidine dimers, (6-4) photophosphates, psoralen-induced damage

-damage recognition -incision (cuts are made on either side of the damage) -excision (removal of the damaged segment) -synthesis of new DNA to replace the excised segment -ligation of new and existing DNA

-defects in NER lead to a disorder called xeroderma pigmentosum (XP) -XP is characterized by severe sensitivity to sunlight, and patients have ~1,000 times greater risk for developing skin cancer -don't have repair mechanisms to repair sun damage

-fixes mispaired bases and short insertions or deletions that occur during DNA replication -DNAPs introduce a mispaired nucleotide every ~10^5 nucleotides ... the 3' --> 5' proofreading exonuclease activity of DNAPs improves fidelity to one mismatch every ~10^7 nucleotides

-the newly synthesized strand contains a mismatch and is not yet methylated (E. coli methylates its DNA at GATC) -newly synthesized DNA is methylated ~2 min after the replication fork passes through -this allows the cell to distinguish old and new DNA -MutS binds to the mismatch and then recruits MutL -the MutS/MutL complex stimulates MutH, which nicks DNA at the GATC site nearest the mismatch -the UvrD helicase unwinds DNA between the nick and the mismatch -single-stranded binding proteins bind the new DNA between the nick and the mismatch preventing it from reannealing to the parental DNA -an exonuclease (which one depends on wehre the nick is relative to the break) chews away the new DNA through the mismatch -DNAPIII fills in the gap and ligase seals the nick -destructive repair mechanism

-discriminating between the old and new strands of DNA in E. coli and other gram-negative bacteria is easy with GATC methylation -eukaryotes can detect mismatches on the lagging strand because they're in Okazaki fragments because they do not have GATC methylation

-during DNA replication, DNAPIII (bacteria) and DNAPs delta and epsilon (eukaryotes) run into lesions that haven't been repaired, like abasic sites or cyclopyrimidine dimers -these lesions can cause DNAPs to fall off DNA -cells use a special class of DNAPs to synthesize DNA across the lesion

error-prone DNA polymerases have a lower fidelity than their replicative counterparts -the plus is that they don't care if the template DNA is damaged, and so the cell can usually survive unrepaired damage -the downside is that these polymerases are more likely to introduce mutations

-a transcriptional response to unrepaired DNA damage -over 40 genes are induced -regulated by two key proteins: LexA and RecA -induces the synthesis of DNA polymerases II, IV, and V in E. coli

-LexA represses the transcription of SOS genes -DNA damage, indicated by a single-stranded region of DNA, is detected by RecA, which forms a filament around the damaged DNA -RecA activates the protease activity of LexA, which cleaves and inactivates itself, allowing translesion polymerases to be expressed

-double strand breaks (DSBs) may be generated through a variety of mechanisms -cells use two major mechanisms to repair DSBs: homologous recombination (uses copy of its chromosome to repair) or nonhomologous end joining (will stick together any free end of DNA --> can lead to mutations)

-DSBs can be generated during DNA replication if there is a nick in one of the template DNA strands -completely dissociates from DNA and causes it to collapse and leads to pausing/stalling of DNA replication -proteins encoded by rec genes are involved in replication via homologous recombination -RecBCD, a helicase complex, unwinds DNA and also has a nuclease activity that leaves a 3' tail on the separated strand of the fork -the 3' tail undergoes "strand invasion" (that is, it displaces a part of the other template strand with the same sequence). strand invasion (causes one strand of unattached DNA to invade duplex) is catalyzed by RecA -the crossed "X" structure created by strand invasion is called a Holliday junction -the Holliday junction migrates (branch migration) via RuvAB. Branch migration influences how much DNA is exchanged between the two strands -RuvC converts the Holliday junction back into a replication fork -PriA loads the DnaB helicase back onto the reestablished replication fork, and replication can keep going

-homologous recombination (HR) is used to repair DSBs in late S and G2 phases of the cell cycle --each chromosome has been replicated and so has a closely associated sister chromatid that can act as a repair template --in theory, the homologous chromosome can be used as a repair template, but this rarely happens because homologous chromosomes are much farther apart than sister chromatids -there are two HR pathways: synthesis-dependent strand annealing (SDSA) and double-strand break repair (DSBR)

-SDSA and DSBR are related pathways, with their early steps being identical -the MRN complex and CtIP binds to the DNA end and chews DNA in a 5' --> 3' direction, leading a single-stranded 3'-OH tail -DNA2 or EXO1 then catalyze more extensive 5' --> 3' degradation

-nonhomologous end joining (NHEJ) is used to repair DSBs when no repair template is available --predominates before DNA replication in G1 and early S phases, before the genome has been replicated and each chromosome has a sister chromatid associated -NHEJ relies on Ku proteins

-Ku and DNA-dependent protein kinase (DNA-PK) are recruited to a DSB -Ku proteins are ring-like and encircle DNA on either side of the DSB -DNA ligase IV attempts to join the broken ends -sometimes, the broken DNA ends are "dirty," lacking either a 5'-phosphate or 3'-OH, and so cannot be immediately ligated -specialized enzymes clean up the dirty DNA ends

-error prone... usually introduces small indels, which can produce protein-altering frameshift mutations -important for generating antibody diversity for the immune system ... does this by joining distinct DNA segments that encode different parts of antibodies

-many human disorders are associated with defects in NHEJ --> LIG4 syndrome or XLF-SCID (severe combined immunodeficiency) -share many features, including cellular sensitivity to radiation, microcephaly (small head), and immunodeficiency