Lecture 10 Flashcards
specific sequence in the genome in which dsDNA is first opened up for replication
origin of replication
most prokaryotes have – per circular genome
one replicon
agrobacteria have linear chromosomes and –
multiple replicons
E. coli – has 245 bp
OriC
OriC has a – followed by –
tandem array of three A-T rich 13-mer regions followed by five 9-mer DnaA binding sites
origin of replication of yeast
ARS1
core sequence of ARS1
A/TTTTAA/GTTTA/T
ori-binding proteins of E. coli
DnaA
ori-binding proteins in yeast
ORC
methylate all E. coli GATC sequence
Dam methylase
methylation of E. coli replication origin creates a – for DNA synthesis initiation
refractory period
approximately – origins of replication are used each time a human cell divides
30,000-50,000
Different cell types use – sets of origin of replications
different
a eukaryotic genome have many origins of replication per –
chromosome
timing of origin activation is related to the –
packing of the local chromatin
dsDNA contains two – but DNA synthesis can only be 5’ to 3’
antiparallel ssDNA
Which of the following is correct for the replication direction?
- 2 origins and 2 growing ends
- 1 origin and 1 fork
- 1 origin and 2 forks
1 origin and 2 forks (bidirectional)
replication bubble gets increasingly longer from both directions but the center is –
constant
one replication bubble has two forks growing in –
the opposite direction
At each replication fork, two ssDNA are replicated in the –
5’ to 3’ direction
labeled replicating DNA show many – fragments being formed (termed Okazaki fragments)
1-2 kb
strand that is synthesized continuously
leading strand
strand that is synthesized discontinuously
lagging strand
– are needed for DNA replication
RNA primers
DNA polymerase can only synthesize DNA if there is –
an existing polynucleotide primer…short RNA sequences
T/F: RNA primers are needed in both prokaryotes and eukaryotes
true
– is the RNA polymerase that synthesizes the short RNA primer
primase
DNA replication is a – process
DNA template-dependent polymerization
replication of sDNA proceeds in – directions
opposite (bidirectional)
DNA replication starts from replication origins that possess –
specific DNA sequences
T/F: each strand of DNA is synthesized continuously and discontinuously
true
required substrates for DNA synthesis
dNTP and primer
DNA is synthesized by extending the – of the primer
3’ end
The DNA catalyzes the – of a deoxyribonucleotide to the 3’ OH end of a polynucleotide chain
stepwise addition
the DNA pol synthesizing reaction is driven by a large, favorable free-energy change cause by the release of – and its subsequent hydrolysis to 2 molecules of inorganic phosphate
pyrophosphate
DNA polymerase has – ability
proofreading
can remove wrong nucleotide one at a time from the ends of polynucleotides
exonuclease
pretty good but not perfect DNA pol proofreading ability is one of the reasons for – in the genome
“spontaneous” mutations
there is about 1 error for every – polymerization event during RNA or protein synthesis
10^4
there is about 1 error for every –polymerization event during DNA replication
10^10
A – suggests why DNA is always synthesized from 5’ to 3’
need for proofreading
helicase binds to – to unwind dsDNA to create ssDNA at the replication fork
replication origins
topoisomerase release supercoil that results from –
DNA unwinding
– stabilize ssDNA before replication
SSB proteins/RPA
primase is a –
DNA dependent RNA polymerase
primase uses – to make short RNA primers
DNA as a template
in eukaryotes, – synthesizes short DNA from the RNA primer, resulting in an RNA-DNA hybrid
DNA pol alpha
eukaryotic primers has – nucleotides
10
one RNA primer is made every – nucleotides on lagging strand
200
DNA ligase connects
backbone of DNA
DNA ligase uses – to activate the 5’ end at the neck before forming the new bond
a molecule of ATP
The energetically unfavorable nick-sealing reaction is driven by being – to the energetically favorable process of ATP hydrolysis
coupled
holds DNA polymerase on the DNA
regulated sliding clamp
ATP binding to clamp loader –>
opens sliding clamp
ATP hydrolysis locks sliding clamp around DNA and – clamp loader
releases
sliding clamps – the DNA polymerase processivity
increase
main synthesizing enzymes in prokaryotes
DNA pol III
main synthesizing enzymes in eukaryotes (lagging)
DNA pol delta
main synthesizing enzymes in eukaryotes (leading)
DNA pol epsilon
prokaryotic clamp and loader
B clamp and gamma clamp loader
eukaryotic clamp and loader
PCNA (proliferating cell nuclear antigen) and RFC (replication factor C)
remove RNA primers in prokaryotes
DNA pol I
remove RNA primers in eukaryotes
RNase H and FEN1
fill in gaps from removed RNA primer in prokaryotes
DNA pol I
fill in gaps from removed RNA primer in eukaryotes
DNA pol delta
connects the Okazaki fragments
DNA ligase
if DNA cannot rapidly rotate –>
torsional stress will build up
some of the tension can be taken up by – whereby the DNA double helix twists around itself
supercoiling
If the tension continues to build up, the replication fork will eventually stop because further unwinding require – energy than the helicase can provide
more
T/F: Topoisomerase I cleave one strand of DNA and ligates the two ends of the broken strand
true
topoisomerase I has – at the active site
tyrosine
DNA topoisomerase – attaches to a DNA phosphate thereby breaking a phosphodiester bond linkage in one strand
covalently
original phosphodiester bond energy is stored in the – making the reaction reversible
phosphotyrosine linkage
– of the phosphodiester bond regenerates both the DNA helix and the DNA topoisomerase
spontaneous reformation
topoisomerase II is used to separate –
2 double helices that are interlocked
topoisomerase II make as reversible covalent attachment to the two strands of one of the double helices creating a double-strand break and forming a –
protein gate
prokaryotic DNA replication begins at the
OriC
around 10 DnaA moleucules bind to the DnaA binding sites which wrap around the DnaA proteins causing the DNA at the – to unpair
13-mer region
denatured 13-mer region recruits
DnaB (helicase) and DnaC (helicase loader) complex
DnaC helps DnaB bind to the – at the 13-mer regino
ssDNA
helicase loading proteins prevent the – from inappropriately entering other single-stranded binding proteins
replicative DNA helices
– inhibit the helices until they are properly loaded at the replication origin
helicase loaders
helicase and primase form
primosome
– of the primase produces the short RNA primers that generate the Okazaki fragment
periodic binding
– is kicked out before DNA pol comes in
SSB proteins
DNA polymerase machinery on each strand work together as part of –
a single complex
the template with the lagging strand – in order for the DNA pol complex to travel in the same direction…towards the replication fork
loops back
prokaryotic helicase
DnaB
eukaryotic helicase
Mcm
prokaryotic helicase loaders
DnaC
eukaryotic helicase loaders
Cdc6 and Cdt1
– stay behind after helices moves on to prevent another round of replication before mitosis is finished
ORC
Mcm helicase + ORC –>
prereplicative complex
an origin of replication can only be used if a – forms in G1 phase
prereplicative complex
At the beginning of S phase, kinases phosphorylate Mcm and ORC, – the former and – the latter
activate Mcm
inactivate ORC
A new pre replicative complex cannot form at the origin until the cell progresses to the next G1 phase, when the bound ORC has been –
dephosphorylated
Mcm helicase moves along the –
leading strand template
bacterial helicase moves along the –
lagging strand template
– synthesizes short RNA-DNA primers on each strand, which mark the starting points to be replicated
primase-DNA pol alpha complex
– replaces the primase-DNA pol alpha complex generating the leading strand
Pol epsilon PCNA-Rfc complex
– synthesize the Okazaki fragment
pol delta PCNA-Rfc complex
RNA-DNA primers are removed by
RNaseH and FEN1
in eukaryotes – fill in gaps with dNTPS
DNA pol delta
the two DNA pol complexes on both strand work together and move –
in the same direction (lagging stand loops back to accommodate)
histones are mainly synthesized in
S phase
physical ends of linear chromosoe
telomeres
3’ end of G rich strand extends – beyond the 5’ end of the complementary C-rich strand
12-16 nucleotides
3’ overhand region is bound by specific proteins that serve to protect the ends of linear chromosomes from
attack by exonuclease
last regions of DNA to be replicated
telomere
– resulting from lagging strand synthesis would become shorter at each cell division
daughter DNA strand
adds telomeric sequences to the ends of each linear chromosome
telomerase
telomerase is a large –
protein-RNA complex
telomerase has – activity that can synthesize DNA from its RNA template
reverse transcriptase
stem cells divide slowly but don’t
shorten telomere
single stranded 3’ end will fold into a T loop with specialized telomere binding proteins – to protect the end of the DNA molecule
shelterin
T/F: most adult somatic cells lack telomerase
true
what has telomerase activity?
stem cells, germ line cells, tumor cells
somatic cells with short telomeres cease dividing
replicative cell senescence
