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Genetics - Lecture > 2.2. DNA Replication > Flashcards

2.2. DNA Replication Flashcards

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1
Q

what are the universal features of replication?

A

(1) both DNA strands as template
(2) semiconservative mode of replication
(3) bidirectional synthesis
(4) 5’ to 3’ direction of synthesis
(5) specific base pairing
(6) requirement for RNA primer

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2
Q

explain how dna is reproduced by semi-conservative replication

A

each strand of the double helix serves as a template for a new strand. when the dna replicates, the two strands separate, and new complementary strands are synthesized for each of the original strands. as a result, each of the two new DNA molecules contains one original strand and one newly sythesized strand.

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3
Q

type of replication wherein the parent DNA molecule remains intact. after replication, one completely new double helix is created and the original helix is conserved

A

conserved replication

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4
Q

type of replication wherein the parental strands are dispersed into two new double helices following replication. hence, each strand consists of both old and new DNA

A

dispersive replication

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5
Q

discuss the meselson-stahl experiment

A

(1) e. coli is initially grown in a medium containing the heavy isotope 15N, resulting in DNA labeled with this heavier isotope
(2) the bacteria are then transferred to a medium with the lighter isotope 14N, causing newly synthesized DNA to incorporate the lighter nitrogen
(3) using cesium chloride density gradient centrifugation, DNA is separated based on density, revealing bands corresponding to different generations of DNA replication.
(4) after one generation, DNA showed a single band of intermediate density, which supports semi-conservative replication; each DNA molecule has one old (heavy) strand and one new (light) strand. however, it also reules out conservative replication, in which two distinct bands would occur
(5) after two cell divisions, DNA showed two bands: one intermediate and one lighter. similar results occurred after a third generation, except that the proportion of the lighter band increased. this was again consistent with the interpretation that replication is semiconservative.

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6
Q

what type of synthesis does DNA follow? explain.

A

bidirectional synthesis : creates two replication forks that move in opposite direction away from the origin of synthesis

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7
Q

formed by the juncture where the two strands separate

A

replication fork

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8
Q

true or false: in bidirectional synthesis, one replication fork is created.

A

false. two replication forks are formed in a bidirectional synthesis

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9
Q

segment of DNA that is replicated as a unit from a single origin

A

replicon

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10
Q

what are the two sites found in a replicon?

A

ori site and termination site

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11
Q

direction of DNA synthesis

A

5’ to 3’

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12
Q

a short segment of RNA complementary to DNA

A

RNA primer

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13
Q

design a 5-bp RNA Primers for the following DNA Sequences:
5’ GGGGCCCTCCTGAACG 3’
3’ CCCCGGGAGGACTTGC 5’

A

primer 1: 5’ CGUUC 3’
primer 2: 5’ GGGGC 3’

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14
Q

differentiate forward primer from reverse primer in polymerase chain reaction (PCR) primers

A

forward primer replicates the 3’ to 5’ template, whereas reverse primer replicates the 5’ to 3’ primer

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15
Q

PCR primer: identify forward and reverse primers given these strands
5’ GGGGCCCTCCTGAACG 3’
3’ CCCCGGGAGGACTTGC 5’

A

forward primer : GGGGC
reverse primer : CGTTC

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16
Q

major DNA replication enzyme in bacteria : replaces supercoils ahead of replisome

A

enzyme : DNA gyrase
encoding genes : gyrAB

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17
Q

major DNA replication enzyme in bacteria : binds origin of replication to open double helix

A

enzyme : origin-binding protein
encoding genes : dnaA

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18
Q

major DNA replication enzyme in bacteria : loads helicase at origin

A

enzyme : helicase loader
encoding genes : dnaC

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19
Q

major DNA replication enzyme in bacteria : unwinds double helix at replication fork

A

enzyme : helicase
encoding genes : dnaB

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20
Q

major DNA replication enzyme in bacteria : prevents single strands from annealing

A

enzyme : SSBP
encoding genes : ssb

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21
Q

major DNA replication enzyme in bacteria : primes new strands of DNA

A

enzyme : primase
encoding genes : dnaG

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22
Q

major DNA replication enzyme in bacteria : main polymerizing enzyme

A

enzyme : DNA polymerase III
encoding genes : n/a

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23
Q

major DNA replication enzyme in bacteria : loads Pol III onto sliding clamp

A

enzyme : clamp loader
encoding genes : holA-E

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24
Q

major DNA replication enzyme in bacteria : holds Pol III on DNA

A

enzyme : sliding clamp
encoding genes : dnaN

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25
Q

major DNA replication enzyme in bacteria : strand elongation

A

enzyme : polymerase subunit
encoding genes : dnaE

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26
Q

major DNA replication enzyme in bacteria : holds together the two core enzymes for the leading and lagging strands

A

enzyme : dimerization subunit (tau)
encoding genes : dnaX

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27
Q

major DNA replication enzyme in bacteria : proofreading

A

enzyme : proofreading subunit
encoding genes : dnaQ

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28
Q

major DNA replication enzyme in bacteria : excises RNA primer and fills in gaps

A

enzyme : DNA polymerase I
encoding genes : polA

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29
Q

major DNA replication enzyme in bacteria : seals nicks in DNA

A

enzyme : DNA ligase
encoding genes : ligA, ligB

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30
Q

major DNA replication enzyme in bacteria : binds terminus and blocks progress of the replication fork

A

enzyme : tus protein
encoding genes : tus

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31
Q

major DNA replication enzyme in bacteria : unlinking of interlocked circles

A

enzyme : topoisomerase IV
encoding genes : parCE

32
Q

large replication complex formed by aggregation of replication proteins

A

replisome

33
Q

function of DNA polymerase I of e.coli

A

(1) dna repair
(2) primer removal
(3) filling of gaps from primer removal

34
Q

function of DNA polymerase III of e.coli

A

primary replication enzyme

35
Q

function of DNA polymerase II, IV, V of e.coli

A

dna repair

36
Q

true or false : DNA polymerases I, II, and III are not capable of initiating chain synthesis

A

true

37
Q

among the three DNA polymerase, which is/are capable of 5’ to 3’ polymerization (addition of nucleotides at the 3’ end)? which is/are capable fo 3’ to 5’ exonuclease activity (proofreading)? which is/are capable of 5’ to 3’ exonuclease activity (removal of RNA primers)?

A

🔸all three are capable fo 5’ to 3’ polymerization and 3’ to 5’ exonuclease activity
🔸only DNA polymerase I can do 5’ to 3’ exonuclease activity or the removal of RNA primers

38
Q

active form of DNA polymerase III which is made up of unique polypeptide subunit, such as α, ε, and θ (there are seven other subunits identified; 10 in total)

A

holoenzyme

39
Q

a complex made up of the largest sub-unit α, along with subunits ε and θ, which imparts the catalytic function to holoenzyme; elongates polynucleotide chain and proofreads

A

core enzyme

40
Q

true or false: in e.coli, each holoenzyme contains two, or possibly three, core enzyme complexes

A

true

41
Q

subunit(s) of DNA Pol III holoenzyme responsible for 5’ to 3’ polymerization

A

α

42
Q

subunit(s) of DNA Pol III holoenzyme responsible for 3’ to 5’ exonuclease activity

A

ε

43
Q

subunit(s) of DNA Pol III holoenzyme responsible for core assembly

A

θ

44
Q

subunit(s) of DNA Pol III holoenzyme responsible for loading enzyme on a template, or the (sliding) clamp loader

A

γ, δ, δ’, χ, ν (known altogether as γ complex)

45
Q

subunit(s) of DNA Pol III holoenzyme responsible for the sliding clamp factor (processivity factor)

A

β

46
Q

subunit(s) of DNA Pol III holoenzyme responsible for dimerizing core complex

A

τ

47
Q

how many different DNA polymerases can be found in humans? in mammalian cells?

A

at least 14; tens of thousands

48
Q

refers to the strength of the association between the enzymes and its substrate, and thus the length of DNA that is synthesized before the enzyme dissociates from the template

A

processivity

49
Q

what are the major eukaryotic DNA polymerases?

A

(1) Pol α
(2) Pol δ
(3) Pol ε

50
Q

eukaryotic DNA pol(s) with low processivity; two of its/their four subunits synthesize RNA primers on both the leading and lagging strands

A

DNA Pol α

51
Q

what happens to DNA Pol α once a primer is put in place?

A

undergoes polymerase switching, whereby Pol α dissociates from the template and is replaced by Pol δ or ε

52
Q

eukaryotic DNA pol(s) with high processivity; extend the primers on opposite strands of DNA; exhibit 3’ to 5’ exonuclease activity, thus having the potential to proofread

A

Pol δ (leading strand) and Pol ε (lagging strand)

53
Q

what are the stages of DNA replication?

A

(1) initiation
(2) elongation
(3) termination

54
Q

in initiation, a protein responsible for initiating replication binds to the origin of replication, which destabilizes and opens up double-stranded DNA, exposing ssDNA regions. what is this protein called?

A

DnaA

DnaA binds to a region of 9mers (found in oriC). this complex then undergoes a slight conformational change and associates with the region of 13mers, causing destabilization

55
Q

site of replication initiation and separation of the double-stranded DNA

A

origin of replication

56
Q

recruits the holoenzyme to bind to the newly formed replication fork and formally initiates replication; unwinds the dsDNA

A

helicase : direction of unwinding is 5’ to 3’

in the pdf : dnaA does the recruiting. in the book, helicase

57
Q

what is needed for helicase to do its job of unwinding the strands?

A

ATP; achieved through hydrolysis

58
Q

prevents single-stranded DNA from annealing

A

single-stranded binding proteins (SSBs); hairpin structures may be formed without SSBs

59
Q

as unwinding proceeds, a coiling tension may be created ahead of the replication fork which produces ____.

A

supercoiling

60
Q

supercoiling may be relaxed by a member of the larger group of enzymes called DNA topoisomerases which makes either single or double-stranded “cuts” and also catalyzes localized movements that have the effect of “undoing” the twists and knots created during supercoiling

A

DNA gyrase

these various reactions (undoing twists and releasing strands after) are driven by the energy released during ATP hydrolysis

61
Q

a short segment of RNA primer which is complementary to DNA is synthesized to achieve a free hydroxyl group. this synthesis is directed by a form of RNA polymerase called ____.

all synthesis follows the 5’ to 3’ direction

A

primase

62
Q

in elongation, this type of DNA pol synthesizes the leading strand continuously in the 5’ to 3’ direction

A

DNA Polymerase III

DNA synthesis is initiated at specific sites along each template strand

63
Q

as the fork progresses, many points of initiation are necessary on the opposite DNA template (since DNA pol synthesizes in 5’ to 3’), resulting in a strand called ___.

A

lagging strand

64
Q

enzymes that excises or removes RNA primers and fills in gaps by replacing missing nucleotides

A

DNA Polymerase I

65
Q

enzyme that seals the nicks in the DNA/join the fragments together by catalyzing the formation of phosphodiester bonds

A

DNA ligase

66
Q

termination begins when the replication fork collides at the ____.

A

terminus of replication

terminus of replication : opposite side of the chromosome from the origin

67
Q

terminus of replication contains specific DNA sequences called ___.

A

Ter sites

68
Q

proteins that recognize ter sites and block progress of the replication forks

A

tus proteins

once the replication fork collides at the terminus of replication, tus proteins bind with ter sites. this tus-ter complex then creates a physical block, which hinders the progress of replication fork and ensures correct termination

69
Q

facilitates DNA partitioning in daughter cells during cell division

A

FtsZ

70
Q

how does proofreading work during DNA replication?

A

(1) proofreading begins at time of nucleotide insertion
(2) mismatched nucleotide is excised from growing DNA strand
(3) correct nucleotide is inserted into growing DNA strand

proofreading increases the fidelity of synthesis by a factor of 100

71
Q

replication through chromatin in eukaryotes

A

(1) removal and modification of nucleosomes and other DNA-binding proteins
(2) tight coupling of DNA synthesis and histone synthesis during S phase
(3) rapid reassociation of histones and non-histone proteins after replication
(4) assembly of new nucleosomes behind the replication fork through chromatin assembly factors (CAFs)

72
Q

problems associated with linear DNA ends of eukaryotes

A

(1) double stranded “ends” of DNA molecules at the termini of linear chromsomes potentially resembles the double-stranded breaks (DSBs) that can occur when a chromosome becomes fragmented internally as a result of DNA damage.
🔸this may be targeted by DNA repair mechanisms
🔸vulnerable to nuclease degradation
(2) during DNA replication, DNA polymerases cannot synthesize new DNA at the tips of single-stranded 5’ ends

73
Q

how are the replication problems at eukaryotic chromosome ends dealt with?

A

addition of nucleotides on telomeres via telomerase

74
Q

region of repetitive DNA sequences at the end of a chromosome. which strand of the DNA is G-rich?

A

telomeres; 3’ strand

75
Q

a ribonucleoprotein enzyme capable of adding several more repeats of the nucleotide sequence to the 3’ end of the G-rich strand

A

telomerase

76
Q

serves as (1) a “guide” to proper attachment of the enzyme to the telomere and (2) a “template” for synthesis of its DNA complement

A

telomerase RNA components (TERC)

77
Q

catalytic subunit of the telomerase enzyme

A

telomerase reverse transcriptase (TERC)

Genetics - Lecture (12 decks)

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