Biochemii Lecture 11,12,13

Front 1

Where does replication begin along a chromosome or plasmid?

Back 1

origin of replication

Front 2

Whose experiments lead to the finding that the replication fork was bi-directional?

Back 2

Cairns

Front 3

In Cairns experiment he observed tritium labeled ___________ was incorporated into ______ loops durind DNA replication in bacteria.

Back 3

thymidine, two

Front 4

In Cairns "pulse-chase" experiments he revealed that ___________ of the replication fork was ____________.

Back 4

movement, bi-directional

Front 5

Which image is correct?

Back 5

the bi-directional one

Front 6

How would you perform a "pulse chase" experiment?

Back 6

In biochemistry and molecular biology, a pulse-chase analysis is a method for examining a cellular process occurring over time by successively exposing the cells to a labeled compound (pulse) and then to the same compound in an unlabeled form (chase). Radioactivity is a commonly used label.

Front 7

Whose experiments concluded that chromosomes or plasmids have an origin of replication?

Back 7

Inman's experiments

Front 8

Inman's experiments used denaturation loops as reference marks on plasmids called ____________ ___________.

Back 8

denaturation mapping

Front 9

Inman concluded that for a given plasmid, replication ____________ at the same place every time. What is this called?

Back 9

originated, point of origin of replication

Front 10

Fill in the blanks.

Back 10

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Fill in the blanks.

Back 11

Front 12

DNA replication requires an unpaired _________ to copy, or what is called a __________ strand.

Back 12

template, parent

Front 13

DNA replication requires a primer to provide a free ______.

Back 13

3' OH

Front 14

DNA replication can add only onto the 3' OH so the strand always grows in a ____________ direction.

Back 14

5'--->3'

Front 15

Draw the DNA polymerase reaction mechanism. Include the amino acids involved and the metal ions.

Back 15

Front 16

The primers used to start DNA synthesis are created by the enzyme ______________>

Back 16

primase

Front 17

Primers created from primase are RNA/DNA?

Back 17

RNA

Front 18

The primers used to start DNA synthesis are small ____ fragments, which are ________ nucleotides in length.

Back 18

RNA, 1-60

Front 19

Primase primers are ultimately (in the end of DNA synthesis) ____________, and replaced with _______.

Back 19

hydorlyzed, DNA

Front 20

Which replication strand needs multiple primers?

Back 20

Lagging strand

Front 21

In E. coli the error rate in DNA synthesis is about 1 per __________ nucleotides incorporated into DNA.

Back 21

10^9-10^10

Front 22

The level of fidelity of E. coli DNA polymerase is insured by the ability of the enzyme to discriminate between __________ and __________ base pairing.

Back 22

correct, incorrect

Front 23

This question is for E. coli DNA polymerase.

Back 23

Front 24

When a nuclease hydrolyzes DNA/RNA it can yield what kind of ends?

Back 24

Front 25

Exonucleases hydrolyze from ____________________ of the nucleic strand.

Back 25

one end or the other

Front 26

Can an exonuclease hydrolyze from either end of the nucleic strand?

Back 26

yes

Front 27

Exonucleases act on _________ strand and ___________ strand DNA/RNA.

Back 27

single, double

Front 28

Endonucleases hydrolyze from ___________ the strand.

Back 28

within

Front 29

Restriction __________ are used in cloning to cut the dsDNA.

Back 29

endonucleases

Front 30

DNA polymerases have nuclease activities for what functions?

Back 30

proofreading function, repair function

Front 31

The proofreading function of DNA polymerases does what?

Back 31

corrects immediate errors in replication by acting as a 3'--->5' exonuclease

Front 32

The repair function of DNA polymerases does what?

Back 32

corrects errors that lie ahead of the replication fork acting as a 5'----->3' exonuclease.

Front 33

The proofreading function and the repair function use what kind of nuclease?

Back 33

exonuclease

Front 34

What strand direction does the proofreading function work?

Back 34

3'--->5'

Front 35

What strand direction does the repair function work?

Back 35

5'---->3'

Front 36

Define nick translation?

Back 36

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Front 42

DNA poly1 is a ___________ polypeptide?

Back 42

single

Front 43

DNA helicase use the energy derived from _____ hydrolysis to _______ and ________ alond dsDNA.

Back 43

ATP, unwind, translocate

Front 44

The DnaB helicase of E. coli is a __________ structure with each subunit containing its own _______ domain.

Back 44

homohexameric, ATPase

Front 45

DNA helicases have a ________ effect, one subunit _______ when bound to ATP, while the next subunit _________ when bound to ADP.

Back 45

rotary
grabs
pulls

Front 46

The rotary effect of a DNA helicase pushes one strand thru the _______, and one strand ________ ________ _______.

Back 46

center of the enzyme, around the outside

Front 47

Coordination between adjacent subunits causes an ATP _________ cycle that pulls ssDNA through the __________ of the enzyme structure.

Back 47

hydrolysis, hole in the center

Front 48

All enzymes and proteins associated with DNA/RNA replication is called the?

Back 48

Replisome

Front 49

This man isolated DNA pol1 from E.coli in 1955.

Back 49

Arthur Kornberg

Front 50

After complete characterization of DNA pol1 it was realized or suggested that DNApol1 __________ be the only DNA polymerase involved in replication.

Back 50

can not

Front 51

What were three characteristics of DNApol1 that Arthur Kornberg stated that led to his suggestion of more than one DNA polymerase?

Back 51

Front 52

What does high and low processivity mean?

Back 52

low processivity means that the polymerase frequently falls off and must rebind to the nucleic acid.

high processivity is a result of not losing this transition time of being off and on the nucleic acid

Front 53

DNA pol3 has how many subunits?

Back 53

10 different types of subunits

Front 54

DNApol3 is considered fast due to high ____________.

Back 54

processivity

Front 55

DNApol3 lacks a nuclease activity, name it.

Back 55

It can proofread, but has no nick repair

Front 56

_____________ is the principle replication DNA polymerase in E.coli.

Back 56

DNApol3

Front 57

Mutations in DNApol1 are __________, but mutations in DNApol3 are __________.

Back 57

unlethal, lethal

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Front 61

What is the functions of the core subunits of DNApol3?

Back 61

polymerization of the nucleotides, 3'-->5' proofreading exonuclease, stabilization of the proofreading subunit

Front 62

What is DnaB?

Back 62

helicase for DNApol3

Front 63

What is the function of the clamp loading complex of DNApol3?

Back 63

Stable template binding, core enzyme dimerization, clamp loader, clamp opener

Front 64

Compare and contrast DNApol1 with DNApol3.

Back 64

Front 65

What is a Klenow fragment?

Back 65

The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. coli is enzymatically cleaved by the protease subtilisin. First reported in 1970,[1] it retains the 5'-3' polymerase activity and the 3’ → 5’ exonuclease activity for removal of precoding nucleotides and proofreading, but loses its 5' → 3' exonuclease activity.

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Front 74

___________ experiments used denaturation mapping and concluded that for a given plasmid, the replication originated at _________________. He then call this ________________.

Back 74

Inman, the same place every time, point of origin

Front 75

In E.coli the specific site of the origin is _______. This site is a ______bp sequence.

Back 75

oriC, 245

Front 76

___________ binds to the oriC DNA sequence causing the DUE site to separate.

Back 76

DnaA-ATP

Front 77

DnaA-ATP bind to the oriC DNA sequence causing an adjacent 45 bp to separate. What is the site where the separation occurs called?

Back 77

DUE DNA unwinding element

Front 78

DnaA-ATP binding at the oriC site occurs only when both strand of ______ sequences are fully ___________________.

Back 78

GATC, methylated at N6 of adenine.

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Front 84

DnaA-ATP binds to R- and I- sites in the origin and induces ___________________.

Back 84

supercoiling

Front 85

____________ binds to R- and I- sites in the origin and induces supercoiling.

Back 85

DnaA-ATP

Front 86

What happens as a result of DnaA-ATP inducing supercoiling in the oriC site?

Back 86

The DUE (DNA unwinding element) separates to relieve the torsional stress.

Front 87

What enzyme binds to and opens the DnaB helicase and loads one enzyme onto each of the separated DUE strands?

Back 87

DnaC-ATP

Front 88

What enzyme generates an RNA primer on each separated strand at the DUE?

Back 88

DnaG primase

Front 89

Which enzyme binds to the loaded DnaB helicases?

Back 89

DNA polymeraseIII holoenzyme

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Front 94

DnaA is active when bound to ______ and inactive when bound to _______.

Back 94

ATP, ADP

Front 95

The full methylation of the GATC sequences at oriC is called?

Back 95

DAM methylation

Front 96

Where on the GATC sequences of oriC does methylatio happen?

Back 96

N6 of adenine

Front 97

___________ is the only phase of DNA replication that is thought to be regulated.

Back 97

Initiation

Front 98

___________ levels are regulated throughout the cell cycle and peak around the start of replication

Back 98

DnaA-ATP

Front 99

DnaA-ATP levels are regulated throughout the cellc ycle and peak around the ___________________.

Back 99

start of replication

Front 100

Loading of the DNA polymerase3 complex stimulates hydrolysis of ___________ to ___________.

Back 100

DnaA-ATP to DnaA-ADP

Front 101

Hydrolysis of DnaA-ATP results in the ______________________. (long answer)

Back 101

disassembly of the DnaA complex at the origin

Front 102

What event stimulates the hydrolysis of DnaA-ATP?

Back 102

the loading of the DNApol3 holoenzyme

Front 103

The conversion of DnaA-ADP back to DnaA-ATP takes a lot of time resulting in?

Back 103

a sufficient lag to allow replication to completeR

Front 104

Replication creates new sites for ___________ binding that lie outside of the origin, effectively titrating away ____________.

Back 104

DnaA-ATP

Front 105

Conversion of the __________________ strands that result from replication back to fully methylated strand takes time.

Back 105

hemimethylated

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The left over nick is repaired by?

Back 117

DNA ligase

Front 118

DNA ligase catalyzes formation of __________ bond by using _______. (only in E.coli)

Back 118

phosphodiester, NAD+

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Name this molecule.

Back 125

NAD+

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