MAMMALIAN GENETICS BCH5413 EXAM 4 LATEST UPDATE

DNA can be both replicated and transcribed into mRNA, which can be translated into protein - ANSWER What is the central dogma of molecular biology? True (this is due to intrastrand crosslinking, in which adjacent nucleotides basepair, causing bubbling) - ANSWER T/F: the addition of bulky aromatic rings and/or exposure to UV light can cause the DNA to "bubble." True - ANSWER T/F: chemotherapy can cause both single and double-stranded breaks in DNA. Damaged DNA can lead to incorrect base pairing, which can lead to mutations (permanent changes in the DNA sequence) - ANSWER Why is it very important that damaged DNA is repaired? Guanine with a new -C=O bond in place of the original =C-H2 bond - ANSWER What is 8-oxyguanine? 8-oxyguanine flips over, causing it to hydrogen bond with adenine (instead of cytosine) and become a thymine in the next round of DNA replication - ANSWER What happens to GC base pairing when guanine is converted to 8-oxyguanine? 2 (instead of guanine's normal 3) - ANSWER How many hydrogen bonds does 8-oxyguanine have with its partner nucleotide? An agent used in chemotherapy that has lots of bulky side chains - ANSWER What is cisplatin? Bulky adducts (DNA "bubbles") caused by the addition of large sidechains and/or intrastrand crosslinking caused by cisplatin binding to 2 adjacent guanines - ANSWER What type of DNA damage can be caused by cisplatin? The addition of cisplatin causes damage to DNA, so introducing it to cancer cells can help damage/kill them - ANSWER Why is cisplatin an effective treatment for cancer? Silent mutation - ANSWER What type of mutation occurs when a single basepair is changed, but the amino acid stays the same? Missense mutation - ANSWER What type of mutation occurs when a single basepair is changed, changing the amino acid? Nonsense mutation - ANSWER What type of mutation occurs when a single basepair is changed, causing the formation of a stop codon? False (mutations in the active site are more detrimental than mutations in outer regions of the protein) - ANSWER T/F: mutations in any part of a protein's sequence can be equally damaging. Frameshift mutation - ANSWER What type of mutation occurs when a single basepair is inserted or deleted? A single-stranded break in the DNA - ANSWER What kind of DNA damage is recognized by the BER repair proteins? Each type of glycosylase recognizes a different type of DNA damage - ANSWER Why is it necessary to have multiple types of glycosylases? Mono-functional glycosylases will only cleave an incorrect base; bi-functional glycosylases will cleave both an incorrect base and the DNA's backbone - ANSWER How does BER differ if a mono-functional vs. a bi-functional glycosylase is used? False ("short-patch" DNA repair is when only 1 base is removed and replaced) - ANSWER T/F: "Short-patch" DNA repair refers to multiple bases being removed and replaced along a short section of DNA. FEN1 (cleaves the "flap" of incorrect bases) - ANSWER What is the additional enzyme that's required for "long-patch" DNA repair before ligase can seal up the nick? (1) To cut the DNA backbone at the 5' end; (2) to proofread for mistakes by DNAP-beta - ANSWER What are the 2 functions of APE1? No ligase has been added, so the fragments are not able to be connected, giving the short band only (21 bp) - ANSWER Functions of APE1 experiment: why is only 1 short band visible in the control lane? DNA ligase is added, causing the 2 fragments to be connected and be located higher on the gel (40 bp) - ANSWER Functions of APE1 experiment: how does the short band become a long band (21 bp - 40 bp)? If the base pairing before a nick is CORRECT, ligase can easily seal it back up; if the base pairing before a nick is a MISMATCH, ligase requires the help of APE1 and DNAP-beta to efficiently seal the nick (low efficiency if ligase acts alone) - ANSWER Functions of APE1 experiment: why does adding ligase to lane 3 (correct A/T pairing) cause robust formation of the long band while adding ligase to lane 8 (G/T mismatch) only results in a small amount of the long band? DNAP-beta is not able to remove the incorrect base pair before replacing it, as it requires APE1 to do so - ANSWER Functions of APE1 experiment: why does the addition of DNAP-beta in lane 9 not improve the efficiency of forming the long band? APE1 is able to remove the mismatched base pair, allowing DNAP-beta to easily add a new base and ligase to seal the nick - ANSWER Functions of APE1 experiment: why does the addition of APE1 in lane 10 improve the efficiency of long band formation? True (ligation efficiency is dependent on APE1 concentration) - ANSWER T/F: the more APE1 added to nicked DNA, the more efficient the ligation is. False (APE1 is required for efficient ligation) - ANSWER T/F: ligation after DNA repair can be efficient without APE1 (only with ligase and DNAP-beta). Since glycosylases target many different types of DNA damage, having certain ones be defective can result in many different consequences - ANSWER Why are there a wide variety of consequences if BER enzymes are defective? Bulky adducts in DNA ("bubbles" caused by intrastrand crosslinking) - ANSWER What kind of DNA damage is recognized by the NER repair proteins? GG-NER is for inactive genes while TC-NER is for genes being actively transcribed - ANSWER What is the basic difference between the GG-NER pathway and the TC-NER pathway? False (oligonucleotide excision involves a long piece of damaged DNA being removed, while "long-patch" DNA repair involves only removing a few bases) - ANSWER T/F: oligonucleotide excision is the same as "long-patch" DNA repair. It uses its beta-sheet "finger" to flip the 2 correct bases (across from the dimerized bases) out of the helix, allowing it to stabilize and act as a scaffold for other proteins - ANSWER GG-NER: how does XPC become stabilized at typical DNA damage by bulky adducts? False (while DNA glycosylases flip out the damaged base for removal, XPC flips out the CORRECT bases that are across from the dimerized bases) - ANSWER T/F: DNA glycosylases and XPC both flip the damaged bases out of the helix for removal. XPC requires the help of DDB (damaged DNA-binding) protein to flip out the correct bases and stabilize - ANSWER GG-NER: how does XPC become stabilized at harder-to-detect DNA damage (extra bulky adducts)? During transcription, RNAP will automatically stall at areas of DNA damage - ANSWER TC-NER: how does the DNA damage get recognized? CSB binds to stalled RNAP and helps recruit both CSA and UVSSA - ANSWER TC-NER: what is the function of CSB in the process of repair? XPF (5' cut) and XPG (3' cut) - ANSWER What 2 proteins cut the oligonucleotide containing the damage in DNA repair? It helps recruit DNAP to synthesize new nucleotides to fill in the gap (where the damaged DNA was removed) - ANSWER What is the role of the sliding clamp (PCNA) in the DNA repair process? XPB (helps open the DNA) and XPD (verifies the DNA damage) - ANSWER Which 2 subunits of helicase are important in opening DNA up for repair and verifying the damage done? If they are unable to repair dimers caused by UV light (NER-defective), they become hypersensitive to UV light - ANSWER Why do all the individuals with NER-associated syndromes exhibit sun sensitivity? Since there are many proteins involved in NER, having defects in any one of them can lead to a different consequence that can manifest in a variety of clinical symptoms - ANSWER Why do the syndromes associated with NER exhibit a wide variety of other symptoms? Base mismatches, insertions, or deletions - ANSWER What kind of DNA damage is recognized by the mismatch mediated repair (MMR) proteins? MutS (alpha + beta) - ANSWER What protein recognizes mismatches, insertions, and deletions in a DNA sequence? MutS uses the nicks in the DNA sequence of the daughter strand to find mismatches (it is the newly synthesized strand, so the template/parent strand won't have any problems - the newly synthesized bases will be where the mismatch is) - ANSWER How does the strand of DNA (parent vs. daughter) help MutS recognize mismatches in DNA?