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DNA Metabolism - Coggle Diagram
DNA Metabolism
Replication in Eukaryotes
Entire genome replicated 1X/cycle
regulation due to cyclin proteins and cyclindependent kinases (CDKs)
cyclins ubiquinated for proteolytic destruction
at the end of the M (mitosis) phase
Initiation of Replication
Requires a prereplicative complex (pre-RCs)
The origin recognition complex (ORC) loads a
helicase onto the DNA.
ORC functions like bacterial DnaA.
Helicase is a hexamer of mini-chromosome
maintenance proteins (MCM2-7).
function like bacterial DnaB helicase.
Rate of Replication
more slowly than E. coli does
Multiple DNA Polymerases in
Eukaryotic Nuclear Replication
DNA Pol α
polymerase/primase activity
not have 3’->5’ proofreading
DNA Pol δ (lagging strand) and DNA Pol ɛ (leading strand) - associated with PCNA (proliferating cell nuclear antigen-protein)
both highly processive
comparable to bacterial DNA Pol III
both 3’->5’ proofreading
Termination of Replication
telomeres are found at the linear ends of the
nuclear chromosomes.
The enzyme telomerase uses RNA
DNA Repair and Mutations
Chemical reactions and some physical processes constantly damage genomic DNA.
The majority are corrected using the undamaged strand as a template.
Accumulation of mutations in eukaryotic cells is strongly correlated with cancer; most carcinogens are also mutagens.
There are thousands of lesions/day (unrepaired DNA damage) but only 1/1,000 become a mutation
Types of DNA Damage
Mismatches
occasional incorporation of
incorrect nucleotides.
Abnormal bases
spontaneous deamination,
chemical alkylation, or exposure to free radicals
Pyrimidine dimers
when DNA is exposed to UV light.
Backbone lesions
exposure to ionizing
radiation and free radicals
DNA Is Synthesized by DNA Polymerases
DNA Elongation Chemistry
Parental DNA strand serves as a template
Nucleoside triphosphates
serve as substrates in strand synthesis
The nucleophilic OH group at the 3’ end
3’-OH is REQUIRED
3’-OH is made a more powerful nucleophile by nearby
Mg2+ ions.
Pyrophosphate (made of the B and γ phosphates) is
a good leaving group
Requires a Primer
Primer = short strand complementary to
the template
contains a 3’-OH
can be made of DNA or RNA (more common)
Features of DNA Polymerase
insertion site
the incoming nucleotide
binds
postinsertion site
the newly made base pair resides when the polymerase
moves forward
DNA Polymerase Can Add
Nucleotides or Dissociate
processivity
The number of nucleotides added before its
dissociation
varies widely from a few nucleotides to many
thousands
Each specific polymerase has its own
processivity and polymerization rate
Initiation of Replication in E. Coli
Begins at the oriC site
Contains highly conserved sequence elements
R sites
form binding site for initiator protein DnaA
A = T-rich region (DNA unwinding element (DUE))
DnaA (I sites)
DnaA Proteins Bind at R and I Sites in oriC
DnaA proteins are ATPases
Eight DnaAs bind to R and I sites.
Associated proteins facilitate DNA bending
DnaB Helicase Continues Initiation
DnaB hexamer structure is opened by the binding of
DnaC
Regulation of Replication Initiation
via Methylation
After replication, oriC is hemimethylated by Dam methylase.
Hemimethylated oriC sequences interact with the
plasma membrane
After a period, oriC sequences are released from membrane.
Dam methylase fully methylates DNA to allow new
DnaA to bind.
Transitioning Between Okazaki Fragments
Core subunits of DNA Pol III dissociate from one B clamp and bind to a new one.
RNA primer is removed by DNA Pol I or Rnase H1
DNA Pol I fills in the gap
DNA ligase seals the backbone
DNA Replication Properties
semiconservative
Each new DNA has one old (parent) strand and one new
(daughter) strand
The Meselson-Stahl Experiment
Bidirectional
circular DNAs with an extra loop
both strands are replicated simultaneously
two replication forks, so bidirectional replication
Synthesis Proceeds in the Direction 5’->3’
The leading strand is made continuously
Elongation of the Leading Strand
Primase (DnaG) makes RNA primer
The DnaG primase interacts with DnaB helicase, but primase moves in the opposite direction to helicase.
DNA Pol III adds nucleotides to the 3’ end of
the strand
The lagging strand is made discontinuously
Okazaki fragments
Elongation of the Lagging Strand
As in leading strand synthesis, primase makes
RNA primer and DNA Pol III adds nucleotides
One asymmetric DNA Pol III dimer complex
synthesizes both strands!
Final Steps in the Synthesis of the Lagging Strand
DNA Is Degraded
Nucleases degrade nucleic acids
DNases degrade only DNA
RNases degrade only RNA
Exonucleases
remove nucleotides from the ends of DNA
3’->5’-exonuclease activity “proofreads”
5’->3’-Exonuclease Activity
Moves ahead of the enzyme, hydrolyzes things in
its path
nick translation
a strand break moves along with enzyme
Endonucleases
cleave bonds within a DNA sequence
Five DNA Polymerases in E. Coli
DNA polymerase I
abundant
not ideal for replication
rate (600 nucleotides/min) is slower than observed
for replication fork movement
low processivity
primary function is in clean-up
5’->3’-Exonuclease
3’->5’-Exonuclease
DNA polymerase III
the principal replication polymerase
Complex structure with 10 types of subunits
The core domains each interact with a dimer of B subunits that increase the processivity of the complex.
form a sliding clamp the prevents dissociation
processivity of DNA Pol III is >500,000 bp because of the B clamps
DNA polymerases II, IV, and V
DNA repair
Origin of Replication
A = T-rich regions ->bubbles
loops always initiate at a unique
origin
DNA Ligase Makes a Bond Betweena 3’-OH and a 5’-PO4
5’-PO4
activated by attachment of
AMP.
3’-OH
displacing AMP
Third Stage of Replication:
Termination
Replication forks meet at a region with 20-bp
sequences Ter
Ter sites found near each other but in opposite
directions
Ter is also a binding site for the protein Tus
causes a replication fork to stop
Geometry of Base Pairing Accounts for High Fidelity
DNA polymerase active site excludes base
pairs with incorrect geometry
BUT DNA polymerases still insert wrong base
Repair mechanisms fix these errors
Requirements for E. Coli DNA Replication
requires over 20 enzymes and proteins.
called the replisome
helicases (use ATP to unwind DNA strands)
topoisomerases (relieve the stress caused by unwinding)
DNA-binding proteins to stabilize separated strands
primases to make RNA primers
DNA ligases to seal nicks between successive nucleotides on
the same strand (i.e., Okazaki fragments)
DNA metabolism consists of a set of tightly regulated processes that achieve these tasks.
