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124 Cards in this Set

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CycD, what are the kinases that act on it? and what is the function of cycD?
CDK4, CDK6 Progression past restriction point at G1/S boundary
CycE and CycA, kinase? Function of cyclin?
CDK2, Initiation of DNA synthesis in early S phase
CycB, cyclin-dependent kinase and function of cyclin?
CDK1, Transition from G2 to M
Cyclin B is _____ and ______ after ________.
tagged, removed, mitosis
Function of T antigen?
helicase
Function of RPA?
Single strand maintenance, similar to SSB in prokaryotes
Funtion of Pol alpha/primase?
Priming
Function of PCNA?
Sliding clamp
Function of RFC?
Clamp loading, it is an ATPase
Function of Pol delta?
Catalysis
Function of MF1?
RNA removal
Function of Ligase I?
Ligation
Function of FEN1?
Nuclease for removal of RNA primers
Function of RNase H1?
Nuclease for removal of RNA primers
Function of Topoisomerase?
Relieves torsional strain due to replication
Function of DNA pol alpha?
Gap filling and synthesis of lagging strand
Function of DNA pol epsilon?
DNA proofreading and repair
Function of DNA pol beta?
DNA repair
Function of DNA pol gamma?
Mitochondrial DNA synthesis
Function of DNA pol delta
Processive, leading strand synthesis
Activities of DNA polymerases?
Selection of correct dNTp, catalyze incorporation, detect mispair and remove by exonuclease activity
Function of telomerase?
replication of telomeric DNA by means of RNA primers and the formation of T-loops
What are the six steps involved in DNA replication in eukaryotes?
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.
What are all the proteins or protein complexes needed for Eukaryotic DNA replication?
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
What phase of interphase is DNA synthesized?
S Phase
type of chromatin that is active
euchromatin
type of chromatin that is inactive, condensed
heterochromatin
Describe the interphase chromatin
DNA is negatively supercoiled loops, loops attached to nuclear matrix
Describe metaphase chromatin
Each chromosome contains proteinaceous scaffold, DNA loops are anchored on scaffold
MAR
Matrix attachment region
SAR
Scaffold Attachment Region
What happens during differentiation?
Nuclei shrink, chromatin mobility decreases
What phase is DNA most compact?
Metaphase
What are the various regions of the chromosome?
centromere, telomere, arms (p and q), banding patterns (that reflect type of chromatin; dark heterochromatic; light euchromatic)
Is constitutive heterochromatin transcribed?
no, never
Centromeric DNA is what type of chromatin?
constitutive heterochromatin
What are the specific structural DNA sequences of telomeres?
Terminal stretches of tandem repeats, Clusters of G's form a planar G quartet, require specialized replication process with telomerases
What is the catalytic subunit of telmorase function?
reverse transcriptase that uses RNA template (template made by pol III)
chromatin consists of:
DNA, protein, RNA
What is the basic unit of the chromatin fiber?
nucleosome
What are the five classes of histones?
H1, H2A, H2B, H3, H4
Where do N-terminal tails exist between?
turns of DNA
The N-terminal tail of histone proteins can be ___,____, or ____.
acetylated, methylated, or phosphorylated
Histones must be _______ to the nucleus to build the _______ fiber, by ___ ___ ___
chaperoned, chromatin, nucleosome assembly protein (NAP1)
What are the 2 steps of chromatin assembly process?
1. Tetramer of H3/H4 deposited on DNA
2. Addition of H2A and H2B
What proteins act as histone chaperones and what do they recognize?
CAF-1p48 subunit or ASF1, specifically acetylated H3/H4
Assembly of nucleosome core on replicating chromatin involves _________.
PCNA (proliferation cell nuclear antigen)
2 models for chromatin remodeling
Pre-emptive model and Dynamic model
What is the evidence for chromatin remodeling?
DNase hypersensitivity reflects changes in chromatin of active genes.
What are the histone modifying enzymes?
Acetylases, deacetylases, methylases, demethylases, kinases
What are two ways to identify Three Eukaryotic RNA Polmerases?
Chromatographic purification
and
α-amanitin sensitivity (poison from mushrooms)
What is the fucntion and location, of RNA pol I, and what does it transcribe?
-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)
What is the function and location of RNA pol II, and what does it transcribe?
-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
What is the function and location of RNA pol III, and what does it transcribe?
-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)
How many subunits does RNA polymerase have?
>10
What do accessory factors primarily recognize, rather than the polymerase itself?
Promoter
What are promoters for RNA pol I and II are mostly upstream of?
Start Site of Transcription
Promoters for RNA pol III CAN lie upstream or downstream?
downstream
Replication is ________ on chromosomal DNA.
bidirectional
_____ is loaded by RFC (Replication Factor C) complex
PCNA
What is the function of T antigen?
helicase
What are two general types of mutations?
point mutations and insertions and deletions mutations
What are the two types of point of mutations? difference?
missense mutation (change in AA to different AA) and nonensense (Change in AA to stop codon)
What are the three types of insertion and deletion mutations? what do they cause?
insertion-frameshift mut.
deletion-frameshift mut.
triplet expansion-disease
_______ is caused by most mutations.
cancer
3 types of DNA repair:
Excision repair- first choice, NER is an example
Double-strand break repair, and damage by-pass
Types of general DNA damage:
Single-base alteration, two-base alteration, chain breaks, cross-linkage
Alkylating agents cause what? fixed by what?
O6-meG, direct repair
spontaneous reactions, radicals, alkylating agents, and x-rays cause what kind of DNA damage? fixed by what?
abasic sites, oxidized, deaminated bases, alkylated bases; base-excision repair (BER)
UV light, environmental mutagens cause what damage to DNA? fixed by what?
pyrimidine dimers and bulky adducts; nucleotide excision repair (NER)
X-rays cause what kind of DNA damage? fixed by what?
double-strand breaks; homologouse recombination (HR), nonhomologous end joining (NHEJ)
replication errors result in what kind of DNA damage? fixed by what?
base mismatches, insertions, deletions; mismatch repair (MMR)
deamination, oxidation, and hydrolysis are all ______ reactions.
spontaneous
what are the common DNA lesions caused by natural processes?
uracil, 8-oxoguanine, thymine glycol, M1G, 1,N6-ethenoadenine, 3-methyladenine, and O6-methylguanine
What are the common DNA lesions caused by environmental (exogenous) agents?
O6-methylguanine, cyclobutane pyrimidine dimers, aromatic amines, nitro compounds, cisplatin
4 mechanisms of DNA repair:
MMR, BER, NER, Double-strand break repair
4 solutions to DNA repair:
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
Spontaneous base loss causes several _____ purines and several _____ pyrimidines to be lossed per haploid genome per day!
thousand, hundred
Loss of a purine or pyrimidine base creates an __________ site.
abasic
CU : known as ______ ______.
spontaneous deamination
other deamination reactions:
adenine to hypoxanthine, guanine to xanthine, 5-methyl cytosine to thymine.
Reactive Oxygen Species (ROS):
O., O-O., HOOH, .OH
A common product of thymine oxidation is _____ _____
thymine glycol
Damage to the DNA backbone leads to what?
nicks
Effects of sunlight:
cyclobutane pyrimidine dimers and pyrimidine dimers
Spontaneous production of 3-methyl adenine caused by what?
S-Adenosylmethionine (SAM)
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?
CPD photolyase, no, DNA ligase
Direct repair of O6-methylguanine adducts by _________-_______
alkylguanyl-transferase (AGT)
All organisms, prokaryotic, eukaryotic, employ at least what three mechanisms of excision repair?
MMR, BER, and NER
MMR, what three enzymes in E. Coli?
MutL, MutH, MutS
MMR process (4 steps):
1. Single strand cut by GATC endonuclease
2. Defect removed by exonuclease
3. Defect repaired by polymerase
4. Religated by ligase
E. Coli-MMR occurs right after _______ and involves ________.
replication, UvrD (helicase II)
MMR in Eukaryotes lacks _____ and _____.
MutH and uvrD
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 ?
PCNA; RPA, Replication factor C (RFC), and DNA polymerase delta.
Hereditary non-polyposis colon cancer (HNPCC) is associated with defects in the genes encoding responsible for ____.
MMR
The "pathway" most commonly employed to removed incorrect bases is called ________.
base excision repair (BER)
What is the common "pathway" for BER?
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.
purpose of DNA N-glycosylase?
cleaves glycosyl bond and leaves an AP site identical to AP site created by spontaneous depurination or depyrimidination.
What enzyme is an enzyme in high # in cancer cells b/c they have higher rates of DNA repair?
glycosylases
6 Steps in NER:
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)
NER in E. Coli, what 3 proteins are responsible for damage recognition and DNA nicking?
UvrA, UvrB, and UvrC
NER in E. Coli, 2 molecules of _____ complex with _______ which is an ATP dependent process.
UvrA, UvrB
What 9-protein complex is particularly noteworthy and essential for DNA reapir and transcription (it stimulates promoter clearing by RNA polymerase II).
TFIIH
Eukaryotic proteins involved in double-strand break repair by homologous recombination:
Rad51, Rad 52, Rad55, Rad57, Rad59, BRCA1 and BRCA2
Homologous recombination is based on the ability of single DNA strands to find regions of near-perfect ______ elsewhere in the genome.
homology
replicons can be linear or _____
circular
How was replicon organization vizualized?
immunofluorescence
Replicon clusters are attached to the nuclear _____.
matrix
During S-Phase, not only does DNA content duplicate but also ______ content.
histone
Origins can be mapped by _______ and _______.
autoradiography and electrophoresis
Replicataion forks create Y-shaped structures that change the electrophoretic ______ of DNA fragments
migration
oriC contains _______ GATC/CTAG repeats that are methylated on adenine on both strands.
eleven
Replication generates _______ DNA, which cannot initiate replication.
hemimethylated
_____ binds to hemimethylated DNA and is required for delaying rereplication. The protein may also interact with _____.
SeqA, DnaA
As origins are hemimethylated, they bind to the cell _______.
membrane
Eukaryotic replicons are ___-___ kb in length
40-100 kb
Individual replicons are activated at characteristic times during _____ phase
S
The ORC is a complex of six proteins that binds to an ____.
ARS
______ ______ is necessary for initiation of replication at each origin.
Licensing factor
What controls the # of times DNA is replicated in eukaryotes?
licensing factor
Licensing factor consists of _____ proteins
MCM
_____ protein is an unstable protein (part of ORC) that is synthesized only in G1.
Cdc6
Cdc6 binds to _____ and allows _______ proteins to bind.
ORC, MCM
_____ _____ maintain mitochondrial origins.
D Loops