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How many replication forks are made at a single ori?
two replication forks
What is ori c recognized by?
a specific initiator protein (DnaA)
What must happen for DnaA ( initiator protein) to act as an initiator?
DnaA must be bound to ATP
What does the binding of DnaA-ATP begin?
A series of events that leads to establishment of replication forks
True or False: Replication initiation is tightly controlled.
true
What is the structure of oriC?
Region is about 250 base pairs long
9-mer sites bind DnaA-ATP—causing the 13-mer (A-T rich DNA) region to melt
What is the process of DNA replication?
DnaA-ATP binds to its recognition sites in OriC
DnaA-ATP nucleoprotein complex causes unwinding of the DNA
DnaC loads DnaB (helicase) onto the ssDNA
Each helicase recruits a primase to initiate DNA synthesis
A clamp loader recognizes the 3’ end and loads a clamp on each strand
The two replisomes are now formed and proceed around the chromosome
Helicase travels on the lagging strand and recruits primase to lay down primers on the lagging strand
What does unwinding of the DNA cause?
local negative supercoiling
How many base pairs is the ori?
about 250 base pairs
What is the recognition site for a DnaA-ATP?
the ori
How does DnaA-ATP form an open complex?
by wrapping around the DNA such as histones would
True or False: There’s a helicase on the leading strand and one on the lagging strand.
true
What knocks DnaA-ATP off the strand?
helicase
What is really knocked off of DnaA-ATP?
a phosphate is knocked off the ATP—slowing down the process
When do tau proteins begin to interact with the helicase?
when a clamp loader recognizes the 3’ end and loads a clamp on each strand
True or False: The primase in each replisome can only prime the lagging strand.
true
True or False: The first lagging strand (Okazaki fragment) serves as the leading strand primer for the other replisome.
true
Why is the leading strand not synthesized as a single continuous molecule?
At some appreciable frequency, the replication fork will “collapse” and need to be reassembled well beyond the origin
True or False: DNA replication must be coordinated with cell division.
true
How is coordination with DNA replication and cell division achieved?
the DNA is methylated
How is DNA methylated?
DAM (DNA adenine methyltransferase) recognizes the sequence and adds a methyl group to the A’s (the methyl group protrudes into the major groove)
True or False: Methylation is slower than replication; it takes time for newly replicated DNA to become methylated.
true
What does methylation tell the cell?
The DNA is newly synthesized
Which strand is the newly synthesized one?
The one that’s not methylated
Which strand is the old one?
The one that is methylated
Hemi-methylated
Will eventually be converted to the fully methylated state
How is replication initiation controlled?
control of DnaA-ATP levels
control of the access to oriC
What is the rate limiting step in initiation?
the binding of DnaA-ATP to OriC
How is the concentration of DnaA-ATP tightly controlled?
The amount of DnaA is proportional to cell mass — small (new) cells do not have enough to initiate replication
As the replication fork passes, DnaA-ATP is converted to DnaA-ADP and it dissociates from its DNA binding sites
DnaA-ADP doesn’t work as an initiator and is very slowly converted to DnaA-ATP
This is one of the ways a new round of initiation is blocked until the time is right
True or False: There are 11 GATC sites of methylation in Ori C.
true
What binds fully methylated DNA?
DnaA-ATP
What binds a hemi-methylated ori c?
the protein Seq A
True or False: Ori C is inactive until fully methylated.
true
True or False: Dam competes with SeqA for binding, but Dam eventually fully methylates the DNA; SeqA slows down methylation but cannot completely block it.
true
When does replication end?
when the two replisomes collide
What does type II topoisomerases do in replication?
decantenation
What is decatenation?
the process of separating interlinked DNA molecules
What does type II topoisomerases do?
Relieves positive supercoils in front of the replication fork
What can replication lead to?
catenated DNA molecules
What resolves the catenane?
the topoisomerase
True or False: Eukaryotic chromosomes have multiple origins.
true
True or False: The replisome of eukaryotes is functionally equivalent to that of E. coli, but the replisome moves more slowly in eukaryotes (30-50 nucleotides/sec).
true
Why does the replisome move slower in eukaryotes?
probably due to chromatin structure
True or False: Humans have a 1000 x more DNA than E. coli, so replication is initiated simultaneously at multiple origins on each chromosome.
true
How far are origins placed apart?
30 kb apart
What is important to know about DNA replication and regulation?
During S phase, all of a eukaryote’s DNA must be replicated once and only once
Incomplete DNA replication leads to chromosomal breaks at cell division
Over-replication can lead to extra copies of regions of chromosomes
True or False: To replicate DNA, eukaryotes have the problem of disassembling all of the structures that compact the DNA into chromatin and, after replication has taken place, putting it all back together.
true
When are helicases loaded and activated?
helicases are loaded at the ori only in G1 and are activated only in the S phase
What is the end replication problem?
The last bit of DNA cannot be synthesized on the Okazaki fragment; it’s chemically impossible
What are telomeres?
the ends of the eukaryotic chromosomes
What do telomeres contain?
tandem repeats of TG rich sequences
What do telomeres do?
this structure protects the ends of linear chromosomes from degradation and other negative things
What is telomerase?
A reverse transcriptase that makes DNA by copying an RNA strand
What does telomerase use as a template?
an internal RNA template
True or False: Telomerase is a ribonucleoprotein.
true
True or False: As with all DNA polymerases, telomerase adds dNTPs to the 3’ end of a primer, but it’s template is RNA and not DNA.
true
Telomerase extends the 3’ end of the telomere so what can happen?
the additional 3’ end DNA can act as template for the new Okazaki fragment, and Okazaki fragment can be repaired
What type of overhang does telomerase form?
3’ overhang
What is senescence?
In higher eukaryotes, most cells don’t express levels of telomerase sufficiently to repair telomeres, so only a limited number of replication/cell division cycles can occur.
What human cells express telomerase and do not sensce?
germ and stem cells
What is the relationship between telomeres and cancer?
Cancer cells divide rapidly and most have acquired mutations that allow expression of telomerase
What is a potential target for anti-cancer drugs?
telomerase
True or False: Telomeres are special and critical for chromosome protection. Also, they contain silenced genes and are bound to the nuclear envelope.
true
