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106 Cards in this Set
- Front
- Back
Funtion of Pol alpha/primase?
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Priming
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Function of PCNA?
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Sliding clamp
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Function of RFC?
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Clamp loading, it is an ATPase
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Function of Pol delta?
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Catalysis
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Function of MF1?
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RNA removal
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Function of Ligase I?
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Ligation
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Function of FEN1?
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Nuclease for removal of RNA primers
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Function of RNase H1?
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Nuclease for removal of RNA primers
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Function of Topoisomerase?
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Relieves torsional strain due to replication
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Function of DNA pol alpha?
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Gap filling and synthesis of lagging strand
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Function of DNA pol epsilon?
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DNA proofreading and repair
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Function of DNA pol beta?
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DNA repair
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Function of DNA pol gamma?
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Mitochondrial DNA synthesis
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Function of DNA pol delta
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Processive, leading strand synthesis
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Activities of DNA polymerases?
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Selection of correct dNTp, catalyze incorporation, detect mispair and remove by exonuclease activity
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Function of telomerase?
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replication of telomeric DNA by means of RNA primers and the formation of T-loops
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What are the six steps involved in DNA replication in eukaryotes?
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1. Indentification of the origins of replication.
2. Unwinding (denaturation) of dsDNA to provide an ssDNA template. 3. Formation of the replication fork. 4. Initiation of replication bubbles with ligation of the newly synthesized DNA segments. 6.Reconstitution of chromatin structure. |
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What are all the proteins or protein complexes needed for Eukaryotic DNA replication?
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ORC, MCM/helicase, CDc2/cyclinB, RPA, PCNA, RFC, Polα, Polδ, Polε, FEN1, Rnase H1, DNA ligase I, T antigen, p21, licensing factor, CDK, DDK, Cdc45p
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What phase of interphase is DNA synthesized?
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S Phase
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type of chromatin that is active
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euchromatin
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type of chromatin that is inactive, condensed
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heterochromatin
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MAR
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Matrix attachment region
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SAR
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Scaffold Attachment Region
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What are the various regions of the chromosome?
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centromere, telomere, arms (p and q), banding patterns (that reflect type of chromatin; dark heterochromatic; light euchromatic)
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Is constitutive heterochromatin transcribed?
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no, never
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Centromeric DNA is what type of chromatin?
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constitutive heterochromatin
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What are the specific structural DNA sequences of telomeres?
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Terminal stretches of tandem repeats, Clusters of G's form a planar G quartet, require specialized replication process with telomerases
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What is the catalytic subunit of telmorase function?
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reverse transcriptase that uses RNA template (template made by pol III)
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chromatin consists of:
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DNA, protein, RNA
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What are the five classes of histones?
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H1, H2A, H2B, H3, H4
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The N-terminal tail of histone proteins can be ___,____, or ____.
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acetylated, methylated, or phosphorylated
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Histones must be _______ to the nucleus to build the _______ fiber, by ___ ___ ___
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chaperoned, chromatin, nucleosome assembly protein (NAP1)
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What are the 2 steps of chromatin assembly process?
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1. Tetramer of H3/H4 deposited on DNA
2. Addition of H2A and H2B |
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What proteins act as histone chaperones and what do they recognize?
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CAF-1p48 subunit or ASF1, specifically acetylated H3/H4
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Assembly of nucleosome core on replicating chromatin involves _________.
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PCNA (proliferation cell nuclear antigen)
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2 models for chromatin remodeling
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Pre-emptive model and Dynamic model
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What is the evidence for chromatin remodeling?
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DNase hypersensitivity reflects changes in chromatin of active genes.
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What are the histone modifying enzymes?
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Acetylases, deacetylases, methylases, demethylases, kinases
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What are two ways to identify Three Eukaryotic RNA Polmerases?
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Chromatographic purification
and α-amanitin sensitivity (poison from mushrooms) |
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What is the fucntion and location, of RNA pol I, and what does it transcribe?
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-Accounts for ~50-70% of total RNA pol activity
-Localized in the nucleolus -Transcribes precursor ribosomal RNA (pre-rRNA 28S, 5.8S, and 18S rRNAs) |
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What is the function and location of RNA pol II, and what does it transcribe?
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-Accounts for ~20-40% of total RNA pol activity
-Localized in the nucleoplasm -Transcribes hetergenous nuclear or pre-messenger RNA; (hnRNA mRNA) plus 4 snRNAs for splicing |
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What is the function and location of RNA pol III, and what does it transcribe?
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-Accounts for ~10% of total RNA pol activity
-Localized in the nucleoplasm -Transcribes transfer RNA (tRNA), 5S rRNA and small stable RNAs (e.g., snRNA & 7S RNA of SRP) |
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How many subunits does RNA polymerase have?
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>10
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What do accessory factors primarily recognize, rather than the polymerase itself?
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Promoter
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What are promoters for RNA pol I and II are mostly upstream of?
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Start Site of Transcription
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Promoters for RNA pol III CAN lie upstream or downstream?
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downstream
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Replication is ________ on chromosomal DNA.
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bidirectional
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_____ is loaded by RFC (Replication Factor C) complex
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PCNA
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What is the function of T antigen?
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helicase
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What are two general types of mutations?
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point mutations and insertions and deletions mutations
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What are the two types of point of mutations? difference?
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missense mutation (change in AA to different AA) and nonensense (Change in AA to stop codon)
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What are the three types of insertion and deletion mutations? what do they cause?
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insertion-frameshift mut.
deletion-frameshift mut. triplet expansion-disease |
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_______ is caused by most mutations.
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cancer
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3 types of DNA repair:
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Excision repair- first choice, NER is an example
Double-strand break repair, and damage by-pass |
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Types of general DNA damage:
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Single-base alteration, two-base alteration, chain breaks, cross-linkage
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Alkylating agents cause what? fixed by what?
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O6-meG, direct repair
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spontaneous reactions, radicals, alkylating agents, and x-rays cause what kind of DNA damage? fixed by what?
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abasic sites, oxidized, deaminated bases, alkylated bases; base-excision repair (BER)
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UV light, environmental mutagens cause what damage to DNA? fixed by what?
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pyrimidine dimers and bulky adducts; nucleotide excision repair (NER)
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X-rays cause what kind of DNA damage? fixed by what?
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double-strand breaks; homologouse recombination (HR), nonhomologous end joining (NHEJ)
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replication errors result in what kind of DNA damage? fixed by what?
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base mismatches, insertions, deletions; mismatch repair (MMR)
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deamination, oxidation, and hydrolysis are all ______ reactions.
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spontaneous
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what are the common DNA lesions caused by natural processes?
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uracil, 8-oxoguanine, thymine glycol, M1G, 1,N6-ethenoadenine, 3-methyladenine, and O6-methylguanine
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What are the common DNA lesions caused by environmental (exogenous) agents?
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O6-methylguanine, cyclobutane pyrimidine dimers, aromatic amines, nitro compounds, cisplatin
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4 mechanisms of DNA repair:
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MMR, BER, NER, Double-strand break repair
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4 solutions to DNA repair:
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MMR: methyl-directed strand cutting, exonuclease digestion, and replacement.
BER: base removal by N-glycosylase, abasic sugar removal, replacement NER: removal of an approximately 30-nucleotide oligomer and replacement. Double-strand break repair: synapsis, unwinding, alignment, ligation |
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Spontaneous base loss causes several _____ purines and several _____ pyrimidines to be lossed per haploid genome per day!
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thousand, hundred
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Loss of a purine or pyrimidine base creates an __________ site.
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abasic
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CU : known as ______ ______.
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spontaneous deamination
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other deamination reactions:
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adenine to hypoxanthine, guanine to xanthine, 5-methyl cytosine to thymine.
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Reactive Oxygen Species (ROS):
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O., O-O., HOOH, .OH
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A common product of thymine oxidation is _____ _____
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thymine glycol
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Damage to the DNA backbone leads to what?
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nicks
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Effects of sunlight:
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cyclobutane pyrimidine dimers and pyrimidine dimers
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Spontaneous production of 3-methyl adenine caused by what?
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S-Adenosylmethionine (SAM)
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Direct reversal of damage is reversible and not kinetically favored but what enzyme can cause photoreversal? is the enzyme found in mammals? What other enzyme can use direct reversal of DNA damage?
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CPD photolyase, no, DNA ligase
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Direct repair of O6-methylguanine adducts by _________-_______
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alkylguanyl-transferase (AGT)
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All organisms, prokaryotic, eukaryotic, employ at least what three mechanisms of excision repair?
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MMR, BER, and NER
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MMR, what three enzymes in E. Coli?
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MutL, MutH, MutS
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MMR process (4 steps):
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1. Single strand cut by GATC endonuclease
2. Defect removed by exonuclease 3. Defect repaired by polymerase 4. Religated by ligase |
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E. Coli-MMR occurs right after _______ and involves ________.
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replication, UvrD (helicase II)
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MMR in Eukaryotes lacks _____ and _____.
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MutH and uvrD
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MMR in eukaryotes; what is required to stabilize MutS and MutL heterodimers at mismatch sites and is also required during the DNA synthesis step of mismatch repair? What other proteins are also required during the DNA synthesis step ?
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PCNA; RPA, Replication factor C (RFC), and DNA polymerase delta.
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Hereditary non-polyposis colon cancer (HNPCC) is associated with defects in the genes encoding responsible for ____.
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MMR
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The "pathway" most commonly employed to removed incorrect bases is called ________.
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base excision repair (BER)
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What is the common "pathway" for BER?
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1. Removal of incorrect base by a DNA N-glycosylase to create AP site.
2. Nicking of the damaged DNA strand by AP endonuclease upstream of the AP site, creating a 3'-OH terminus adjacent to the AP site. 3. Extension of the 3'-OH terminus by a DNA polymerase, accompanied by the excision of the AP site. |
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purpose of DNA N-glycosylase?
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cleaves glycosyl bond and leaves an AP site identical to AP site created by spontaneous depurination or depyrimidination.
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What enzyme is an enzyme in high # in cancer cells b/c they have higher rates of DNA repair?
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glycosylases
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6 Steps in NER:
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1. Damage recognition
2. Binding of a multi-protein complex at damaged site. 3. Double incision of the damaged strand several nucleotides away from the damaged site, on both the 5' and 3' sides 4. Removal of the damage-containing oligonucleotide from between the two nicks. 5. Filling in of the resulting gap by a DNA polymerase 6. Ligation (ligase) |
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NER in E. Coli, what 3 proteins are responsible for damage recognition and DNA nicking?
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UvrA, UvrB, and UvrC
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NER in E. Coli, 2 molecules of _____ complex with _______ which is an ATP dependent process.
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UvrA, UvrB
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What 9-protein complex is particularly noteworthy and essential for DNA reapir and transcription (it stimulates promoter clearing by RNA polymerase II).
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TFIIH
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Eukaryotic proteins involved in double-strand break repair by homologous recombination:
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Rad51, Rad 52, Rad55, Rad57, Rad59, BRCA1 and BRCA2
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Homologous recombination is based on the ability of single DNA strands to find regions of near-perfect ______ elsewhere in the genome.
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homology
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replicons can be linear or _____
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circular
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How was replicon organization vizualized?
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immunofluorescence
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Replicon clusters are attached to the nuclear _____.
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matrix
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During S-Phase, not only does DNA content duplicate but also ______ content.
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histone
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Origins can be mapped by _______ and _______.
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autoradiography and electrophoresis
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Replicataion forks create Y-shaped structures that change the electrophoretic ______ of DNA fragments
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migration
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oriC contains _______ GATC/CTAG repeats that are methylated on adenine on both strands.
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eleven
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Replication generates _______ DNA, which cannot initiate replication.
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hemimethylated
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_____ binds to hemimethylated DNA and is required for delaying rereplication. The protein may also interact with _____.
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SeqA, DnaA
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As origins are hemimethylated, they bind to the cell _______.
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membrane
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Eukaryotic replicons are ___-___ kb in length
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40-100 kb
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Individual replicons are activated at characteristic times during _____ phase
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S
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The ORC is a complex of six proteins that binds to an ____.
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ARS
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