Dna Replication In Eukaryotes

Front 1

CycD, what are the kinases that act on it? and what is the function of cycD?

Back 1

CDK4, CDK6 Progression past restriction point at G1/S boundary

Front 2

CycE and CycA, kinase? Function of cyclin?

Back 2

CDK2, Initiation of DNA synthesis in early S phase

Front 3

CycB, cyclin-dependent kinase and function of cyclin?

Back 3

CDK1, Transition from G2 to M

Front 4

Cyclin B is _____ and ______ after ________.

Back 4

tagged, removed, mitosis

Front 5

Function of T antigen?

Back 5

helicase

Front 6

Function of RPA?

Back 6

Single strand maintenance, similar to SSB in prokaryotes

Front 7

Funtion of Pol alpha/primase?

Back 7

Priming

Front 8

Function of PCNA?

Back 8

Sliding clamp

Front 9

Function of RFC?

Back 9

Clamp loading, it is an ATPase

Front 10

Function of Pol delta?

Back 10

Catalysis

Front 11

Function of MF1?

Back 11

RNA removal

Front 12

Function of Ligase I?

Back 12

Ligation

Front 13

Function of FEN1?

Back 13

Nuclease for removal of RNA primers

Front 14

Function of RNase H1?

Back 14

Nuclease for removal of RNA primers

Front 15

Function of Topoisomerase?

Back 15

Relieves torsional strain due to replication

Front 16

Function of DNA pol alpha?

Back 16

Gap filling and synthesis of lagging strand

Front 17

Function of DNA pol epsilon?

Back 17

DNA proofreading and repair

Front 18

Function of DNA pol beta?

Back 18

DNA repair

Front 19

Function of DNA pol gamma?

Back 19

Mitochondrial DNA synthesis

Front 20

Function of DNA pol delta

Back 20

Processive, leading strand synthesis

Front 21

Activities of DNA polymerases?

Back 21

Selection of correct dNTp, catalyze incorporation, detect mispair and remove by exonuclease activity

Front 22

Function of telomerase?

Back 22

replication of telomeric DNA by means of RNA primers and the formation of T-loops

Front 23

What are the six steps involved in DNA replication in eukaryotes?

Back 23

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.

Front 24

What are all the proteins or protein complexes needed for Eukaryotic DNA replication?

Back 24

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

Front 25

What phase of interphase is DNA synthesized?

Back 25

S Phase

Front 26

type of chromatin that is active

Back 26

euchromatin

Front 27

type of chromatin that is inactive, condensed

Back 27

heterochromatin

Front 28

Describe the interphase chromatin

Back 28

DNA is negatively supercoiled loops, loops attached to nuclear matrix

Front 29

Describe metaphase chromatin

Back 29

Each chromosome contains proteinaceous scaffold, DNA loops are anchored on scaffold

Front 30

MAR

Back 30

Matrix attachment region

Front 31

SAR

Back 31

Scaffold Attachment Region

Front 32

What happens during differentiation?

Back 32

Nuclei shrink, chromatin mobility decreases

Front 33

What phase is DNA most compact?

Back 33

Metaphase

Front 34

What are the various regions of the chromosome?

Back 34

centromere, telomere, arms (p and q), banding patterns (that reflect type of chromatin; dark heterochromatic; light euchromatic)

Front 35

Is constitutive heterochromatin transcribed?

Back 35

no, never

Front 36

Centromeric DNA is what type of chromatin?

Back 36

constitutive heterochromatin

Front 37

What are the specific structural DNA sequences of telomeres?

Back 37

Terminal stretches of tandem repeats, Clusters of G's form a planar G quartet, require specialized replication process with telomerases

Front 38

What is the catalytic subunit of telmorase function?

Back 38

reverse transcriptase that uses RNA template (template made by pol III)

Front 39

chromatin consists of:

Back 39

DNA, protein, RNA

Front 40

What is the basic unit of the chromatin fiber?

Back 40

nucleosome

Front 41

What are the five classes of histones?

Back 41

H1, H2A, H2B, H3, H4

Front 42

Where do N-terminal tails exist between?

Back 42

turns of DNA

Front 43

The N-terminal tail of histone proteins can be ___,____, or ____.

Back 43

acetylated, methylated, or phosphorylated

Front 44

Histones must be _______ to the nucleus to build the _______ fiber, by ___ ___ ___

Back 44

chaperoned, chromatin, nucleosome assembly protein (NAP1)

Front 45

What are the 2 steps of chromatin assembly process?

Back 45

1. Tetramer of H3/H4 deposited on DNA
2. Addition of H2A and H2B

Front 46

What proteins act as histone chaperones and what do they recognize?

Back 46

CAF-1p48 subunit or ASF1, specifically acetylated H3/H4

Front 47

Assembly of nucleosome core on replicating chromatin involves _________.

Back 47

PCNA (proliferation cell nuclear antigen)

Front 48

2 models for chromatin remodeling

Back 48

Pre-emptive model and Dynamic model

Front 49

What is the evidence for chromatin remodeling?

Back 49

DNase hypersensitivity reflects changes in chromatin of active genes.

Front 50

What are the histone modifying enzymes?

Back 50

Acetylases, deacetylases, methylases, demethylases, kinases

Front 51

What are two ways to identify Three Eukaryotic RNA Polmerases?

Back 51

Chromatographic purification
and
α-amanitin sensitivity (poison from mushrooms)

Front 52

What is the fucntion and location, of RNA pol I, and what does it transcribe?

Back 52

-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)

Front 53

What is the function and location of RNA pol II, and what does it transcribe?

Back 53

-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

Front 54

What is the function and location of RNA pol III, and what does it transcribe?

Back 54

-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)

Front 55

How many subunits does RNA polymerase have?

Back 55

>10

Front 56

What do accessory factors primarily recognize, rather than the polymerase itself?

Back 56

Promoter

Front 57

What are promoters for RNA pol I and II are mostly upstream of?

Back 57

Start Site of Transcription

Front 58

Promoters for RNA pol III CAN lie upstream or downstream?

Back 58

downstream

Front 59

Replication is ________ on chromosomal DNA.

Back 59

bidirectional

Front 60

_____ is loaded by RFC (Replication Factor C) complex

Back 60

PCNA

Front 61

What is the function of T antigen?

Back 61

helicase

Front 62

What are two general types of mutations?

Back 62

point mutations and insertions and deletions mutations

Front 63

What are the two types of point of mutations? difference?

Back 63

missense mutation (change in AA to different AA) and nonensense (Change in AA to stop codon)

Front 64

What are the three types of insertion and deletion mutations? what do they cause?

Back 64

insertion-frameshift mut.
deletion-frameshift mut.
triplet expansion-disease

Front 65

_______ is caused by most mutations.

Back 65

cancer

Front 66

3 types of DNA repair:

Back 66

Excision repair- first choice, NER is an example
Double-strand break repair, and damage by-pass

Front 67

Types of general DNA damage:

Back 67

Single-base alteration, two-base alteration, chain breaks, cross-linkage

Front 68

Alkylating agents cause what? fixed by what?

Back 68

O6-meG, direct repair

Front 69

spontaneous reactions, radicals, alkylating agents, and x-rays cause what kind of DNA damage? fixed by what?

Back 69

abasic sites, oxidized, deaminated bases, alkylated bases; base-excision repair (BER)

Front 70

UV light, environmental mutagens cause what damage to DNA? fixed by what?

Back 70

pyrimidine dimers and bulky adducts; nucleotide excision repair (NER)

Front 71

X-rays cause what kind of DNA damage? fixed by what?

Back 71

double-strand breaks; homologouse recombination (HR), nonhomologous end joining (NHEJ)

Front 72

replication errors result in what kind of DNA damage? fixed by what?

Back 72

base mismatches, insertions, deletions; mismatch repair (MMR)

Front 73

deamination, oxidation, and hydrolysis are all ______ reactions.

Back 73

spontaneous

Front 74

what are the common DNA lesions caused by natural processes?

Back 74

uracil, 8-oxoguanine, thymine glycol, M1G, 1,N6-ethenoadenine, 3-methyladenine, and O6-methylguanine

Front 75

What are the common DNA lesions caused by environmental (exogenous) agents?

Back 75

O6-methylguanine, cyclobutane pyrimidine dimers, aromatic amines, nitro compounds, cisplatin

Front 76

4 mechanisms of DNA repair:

Back 76

MMR, BER, NER, Double-strand break repair

Front 77

4 solutions to DNA repair:

Back 77

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

Front 78

Spontaneous base loss causes several _____ purines and several _____ pyrimidines to be lossed per haploid genome per day!

Back 78

thousand, hundred

Front 79

Loss of a purine or pyrimidine base creates an __________ site.

Back 79

abasic

Front 80

CU : known as ______ ______.

Back 80

spontaneous deamination

Front 81

other deamination reactions:

Back 81

adenine to hypoxanthine, guanine to xanthine, 5-methyl cytosine to thymine.

Front 82

Reactive Oxygen Species (ROS):

Back 82

O., O-O., HOOH, .OH

Front 83

A common product of thymine oxidation is _____ _____

Back 83

thymine glycol

Front 84

Damage to the DNA backbone leads to what?

Back 84

nicks

Front 85

Effects of sunlight:

Back 85

cyclobutane pyrimidine dimers and pyrimidine dimers

Front 86

Spontaneous production of 3-methyl adenine caused by what?

Back 86

S-Adenosylmethionine (SAM)

Front 87

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?

Back 87

CPD photolyase, no, DNA ligase

Front 88

Direct repair of O6-methylguanine adducts by _________-_______

Back 88

alkylguanyl-transferase (AGT)

Front 89

All organisms, prokaryotic, eukaryotic, employ at least what three mechanisms of excision repair?

Back 89

MMR, BER, and NER

Front 90

MMR, what three enzymes in E. Coli?

Back 90

MutL, MutH, MutS

Front 91

MMR process (4 steps):

Back 91

1. Single strand cut by GATC endonuclease
2. Defect removed by exonuclease
3. Defect repaired by polymerase
4. Religated by ligase

Front 92

E. Coli-MMR occurs right after _______ and involves ________.

Back 92

replication, UvrD (helicase II)

Front 93

MMR in Eukaryotes lacks _____ and _____.

Back 93

MutH and uvrD

Front 94

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 ?

Back 94

PCNA; RPA, Replication factor C (RFC), and DNA polymerase delta.

Front 95

Hereditary non-polyposis colon cancer (HNPCC) is associated with defects in the genes encoding responsible for ____.

Back 95

MMR

Front 96

The "pathway" most commonly employed to removed incorrect bases is called ________.

Back 96

base excision repair (BER)

Front 97

What is the common "pathway" for BER?

Back 97

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.

Front 98

purpose of DNA N-glycosylase?

Back 98

cleaves glycosyl bond and leaves an AP site identical to AP site created by spontaneous depurination or depyrimidination.

Front 99

What enzyme is an enzyme in high # in cancer cells b/c they have higher rates of DNA repair?

Back 99

glycosylases

Front 100

6 Steps in NER:

Back 100

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)

Front 101

NER in E. Coli, what 3 proteins are responsible for damage recognition and DNA nicking?

Back 101

UvrA, UvrB, and UvrC

Front 102

NER in E. Coli, 2 molecules of _____ complex with _______ which is an ATP dependent process.

Back 102

UvrA, UvrB

Front 103

What 9-protein complex is particularly noteworthy and essential for DNA reapir and transcription (it stimulates promoter clearing by RNA polymerase II).

Back 103

TFIIH

Front 104

Eukaryotic proteins involved in double-strand break repair by homologous recombination:

Back 104

Rad51, Rad 52, Rad55, Rad57, Rad59, BRCA1 and BRCA2

Front 105

Homologous recombination is based on the ability of single DNA strands to find regions of near-perfect ______ elsewhere in the genome.

Back 105

homology

Front 106

replicons can be linear or _____

Back 106

circular

Front 107

How was replicon organization vizualized?

Back 107

immunofluorescence

Front 108

Replicon clusters are attached to the nuclear _____.

Back 108

matrix

Front 109

During S-Phase, not only does DNA content duplicate but also ______ content.

Back 109

histone

Front 110

Origins can be mapped by _______ and _______.

Back 110

autoradiography and electrophoresis

Front 111

Replicataion forks create Y-shaped structures that change the electrophoretic ______ of DNA fragments

Back 111

migration

Front 112

oriC contains _______ GATC/CTAG repeats that are methylated on adenine on both strands.

Back 112

eleven

Front 113

Replication generates _______ DNA, which cannot initiate replication.

Back 113

hemimethylated

Front 114

_____ binds to hemimethylated DNA and is required for delaying rereplication. The protein may also interact with _____.

Back 114

SeqA, DnaA

Front 115

As origins are hemimethylated, they bind to the cell _______.

Back 115

membrane

Front 116

Eukaryotic replicons are ___-___ kb in length

Back 116

40-100 kb

Front 117

Individual replicons are activated at characteristic times during _____ phase

Back 117

S

Front 118

The ORC is a complex of six proteins that binds to an ____.

Back 118

ARS

Front 119

______ ______ is necessary for initiation of replication at each origin.

Back 119

Licensing factor

Front 120

What controls the # of times DNA is replicated in eukaryotes?

Back 120

licensing factor

Front 121

Licensing factor consists of _____ proteins

Back 121

MCM

Front 122

_____ protein is an unstable protein (part of ORC) that is synthesized only in G1.

Back 122

Cdc6

Front 123

Cdc6 binds to _____ and allows _______ proteins to bind.

Back 123

ORC, MCM

Front 124

_____ _____ maintain mitochondrial origins.

Back 124

D Loops