DNA Replication Flashcards
Explain the fundamental rules of DNA replication
DNA Replication is Semi conservative: Each DNA strand serves as a
template to form 2 new DNA molecules, each with a parent strand and a
new strand.
Replication beings at an Origin and usually proceeds bidirectionally.
Replication forks are formed where the parent DNA are unwound and
the separated strands are quickly replicated.
DNA synthesis occurs in the 5’ 3’ direction and is continuous along
one strand and discontinuous along other strand.
What are the stages of DNA replication?
Initiation
Elongation
Termination
Describe the initiation phase of DNA replication
Initiation:
Interaction of proteins with the origin of replication results in local
unwinding of DNA at an adjacent A=T rich site.
DNA in this area is stabilized by single-strand binding proteins (SSB).
This allows helicase, primase & DNA polymerase to bind and initiate
DNA synthesis.
Only phase to be regulated, mechanism is not fully understood.
Describe the elongation phase of replication
Elongation:
Replication fork is formed and proceeds as DNA synthesis occurs
- continuously on the leading strand
- discontinuously on the lagging strand
The selection of new deoxyribonucleotides to be attached is dependent upon proper
base pairing which is dictated by the template strand.
New nucleotides are added to the free 3’hydroxyl group of the primer
by phosphodiester bonds which is facilitated by Mg²⁺ ions.
Describe the termination phase of replication
Termination:
In circular E.coli chromosome, the 2 replication forks meet at a terminus
region containing multiple copies of a 20 bp sequence called Ter.
In linear eukaryotic chromosomes, termination is brought about by
synthesis of special sequences called telomeres at the end of the
chromosomes.
Explain the properties that all DNA polymerases share
1) Chain elongation:
DNA polymerase adds nucleotide to new DNA strand.
The nucleophile is the 3’ hydroxyl group of the nucleotide at the 3’
end of the growing strand. Nucleophilic attack occurs at the α
phosphate of the incoming nucleotide. Inorganic phosphate is
released.
2) Processivity is defined by the average number of nucleotide added
before a polymerase dissociates.
3) Proofreading identifies copying errors and corrects them, the 3’ 5’
exonuclease activity of polymerase removes mispaired nucleotide.
The exonuclease activity of polymerase double checks after each
nucleotide is added. It is highly specific for mismatched base pairs.
If wrong nucleotide has been added, translocation of polymerase to
the position where the next nucleotide is to be added is halted. The
error is corrected before polymerase begins again
List the claases of proteins involved in replication and state their functions
- DNA polymerases – deoxynucleotide polymerization
- Helicases – processive unwinding of DNA
- Topoisomerases – relieve torsional strain that results from helicase-induced unwinding
- DNA primase – initiates synthesis of RNA primers
- SSB proteins – prevent premature reannealing of dsDNA
- DNA ligase – seals the single strand nick between the nascent chain and Okazaki fragments on lagging strand
Describe the funcions of DNA polymerase I
1) DNA polymerase I: It possesses four enzymatic activites;
A 5’ -> 3’ (forward) DNA-dependent DNA polymerase activity, requiring
a 3’ primer site and a DNA template strand.
A 3’ -> 5’ (reverse) exonuclease activity that mediates proofreading.
A 5’ -> 3’ (forward) exonuclease activity mediating nick
translation during DNA repair.
A 5’ -> 3‘ (forward) RNA-dependent DNA polymerase activity.
Polymerase I operates on RNA templates with considerably lower
efficiency (0.1–0.4%) than it does on DNA templates, and this activity is
probably of only limited biological significance
What is the function of DNA polymerase II?
DNA polymerase II: This enzyme is involved in DNA repair. It differs
from polymerase I in that it lacks a 5’->3’ exonuclease activity
Describe the DNA polymerase III complex with all its subunits
DNA polymerase III: This enzyme is more complex with ten subunits.
Its polymerization and proofreading properties reside in α and ε
subunits, respectively.
θ subunit associates with α and ε to form a core polymerase, which
can polymerize DNA but with limited processivity.
Two core polymerases are linked to another set of subunits, a clamploading complex or γ-complex, consisting of five subunits of four types
τ₂γδδ’.
The core polymerases are linked through the τ (tau) subunit.
Two additional subunits χ (chi) and ψ (psi) are bound to γ-complex.
The increase in processivity of polymerase III is provided by the addition
of the β subunits, four of which complete the DNA polymerase III
holoenzyme
The β subunits associates in pairs to form donut shaped structures that
encircle the DNA and acts like clamp.
The β sliding clamp prevents dissociation of polymerase III from DNA
increasing processivity.
What is the function of DNA polymerase IV & V?
DNA polymerase IV & DNA polymerase V: Involved in unusual form of
DNA repair
Explain the initiation phase of e. coli replication
E.coli replication origin, oriC, consists of 245 bp. The key sequences of interest here are 2 series of short repeats: 3 repeats of 13 bp and 4 repeats of 9 bp.
About 20 DnaA protein molecules, each with a bound ATP, bind at the four 9 bp repeats.
The 3 A=T rich 13 bp repeats are denatured sequentially.
Two ring shaped hexamers of the DnaB proteins binds to the denatured region with the aid of DnaC protein.
The DnaB helicase activity further unwinds the DNA bidirectionally creating two replication forks for the priming by DnaG primase and DNA synthesis.
Many SSB stabilizes the separated strands pre- venting renaturation
DNA gyrase (topoisomerase) relieves the topological stress produced by DnaB helicase.
The timing of replication initiation is
affected by DNA methylation.
The oriC DNA is methylated by Dam
methylase (Dam: DNA adenine methylation)
which methylates the N⁶ position of adenine within (5’) GATC palindromic sequence.
Immediately after replication, DNA is hemi-methylated: parent strand has methylated oriC and the new strand does not.
After a time, the new DNA is fully methylated by Dam methylase and only then can it again bind to DnaA.
Initiation is also regulated by slow hydrolysis of ATP by Dna A
Explain the elongation phase of e. coli replication
Elongation phase includes two distinct but related processes: leading strand synthesis and lagging strand synthesis.
Leading strand synthesis is the straightforward of the two, begins with
synthesis by DnaG primase of a short RNA primer at the replication origin.
Deoxyribonucleotides are added to the primer by DNA polymerase III.
Leading strand synthesis proceeds continuously keeping up with the
unwinding of the DNA at the replication fork.
Lagging strand synthesis is discontinuous and accomplished in short Okazaki fragments.
First, as in leading strand synthesis, primer is synthesized by primase, then DNA polymerase III binds to the RNA primer and adds deoxyribonucleotides.
In order to coordinate the leading and lagging strand synthesis, both strands are produced by a single asymmetrical DNA polymerase III dimer.
This is accomplished by looping the lagging strand so that DNA synthesis proceeds steadily and at the same time on both leading and lagging strand templates
β subunit of DNA polymerase III acts as a sliding clamp which positions at the primer by the clamp-loading complex of DNA polymerase III.
When the synthesis of an Okazaki fragment has been completed, replication halts, and the core subunits of polymerase III dissociates
from the β sliding clamp and the completed Okazaki fragment, and associates with the new clamp.
This initiates synthesis of new Okazaki fragment.
Once an Okazaki fragment has been completed, its RNA primer is removed by 5’ 3’ exonuclease activity of DNA polymerase I and
replaced by DNA by same enzyme.
The remaining nick is sealed by DNA ligase.
DNA ligase catalyzes the formation of phosphodiester bonds between 3’
hydroxyl end of one strand and the 5’ phosphate group at the end of another strand.
The entire replication complex responsible for coordinated DNA synthesis at a replication fork is called a replisome. Replisome brings
about rapid DNA synthesis adding ~1000 nucleotides/sec.
The helicase and primase constitute a functional unit within the replication complex called primosome. The helicase associates with primase to afford the latter proper access to the template
Explain the termination phase of e. coli replication
Eventually the two replication forks of the circular E.coli chromosome
meet at a terminus region called Ter containing multiple copies of a 20
bp sequence.
The Ter functions as a binding site for a protein called Tus (terminus
utilization substance).
The Ter-Tus complex can arrest a replication fork from only one
direction.
Only one Ter-Tus complex function per replication cycle.
Replication generally halts when opposing
replication forks collide, but in case one
replication fork is delayed or halted by
encountering DNA damage or other
obstacles, Ter-Tus may prevent overreplication by other replication fork.
So when a replication fork encounters
Ter-Tus complex it halts, and other fork halts when it meets the first fork.
The final few hundred base pairs of DNA between these two complexes
are then replicated, completing two topologically interlinked circular
chromosomes, known as catenanes.
Separation of the catenated chromosomes require topoisomerase IV.
The separated chromosomes then segregate into daughter cells after
cell division.
How many bp are there in e. coli replication? State their repeats
E.coli replication origin, oriC, consists of 245 bp. The
key sequences of interest here are 2 series of short
repeats: 3 repeats of 13 bp and 4 repeats of 9 bp
