FCH 532 Lecture 13
Chapter 30: DNA replication
Primase
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Closely associated with DNA helicase (T7 gene 4
helicase/primasehas both domains)
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E. coli primase (DnaG) forms noncovalent complex with
DnaB
Primase reverses its direction in order to synthesize RNA
primer in 5’-3’ direction.
•
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3 domains: N-terminal Zn2+ binding domain, central
catalytic domain with Mg2+ , C-terminal domain interacts
with DnaB.
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Figure 30-22 X-Ray structure of E. coli primase.
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DNA replication in E. coli
• Chromosome replicates bidirectionally from a single
replication origin.
• Replisome-active complex that synthesizes both the
leading and lagging strands. Contains 2 Pol III core
enzymes () that are bound to  dimer that
connects the  subunits
  dimer also binds to DnaB (helicase)
DNA replication in E. coli initiated at oriC
•
oriC - Unique 245-bp segment that is highly conserved in gram-negative
bacteria-supports bidirectional replication.
DnaA protein-467 aa, recognizes and binds oriC’s DnaA boxes (highly
conserved 9 bp sequences)
1.
–
–
–
(5’-TTATCCACA-3’)
Forms negative supercoiled DNA wrapped around 5 DnaA proteins.
Facilitated by HU and integration host factor (IHF) that cause bending of
DNA.
DnaA melts 3-tandemly repeated 13 bp AT-rich segments, 5’GATCTNTTNTTTT-3’ located near the left boundry.
2.
•
3.
4.
Established by P1 nuclease
DnaA recruits 2 DnaB6-DnaC6 complexes to form prepriming complex.
SSB and gyrase, DnaB helixas further unwinds the DNA in prepriming
complex in both directions
•
oriC sequence similar to RNA promoters (AT-rich) and RNAP activates
primase for production RNA primers
Figure 30-29 A model for DNA
replication initiation at oriC.
1. DnaA proteins bind to DnaA
boxes at oriC aided by HU
and IHF
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2. 3 AT-rich 13-bp repeats are
melted in ATP dependent
fashion-open complex
3. 2 DnaB6-DnaC6 complexes
recruited to opposite ends of
complex + 5 DnaA to form
dimers.
4. Open complex further
unwound by DnaB
Regulation of initiation of DNA replication
The doubling time of E. coli at 37 °C varies from <20 min to 10 h.
Replication fork has a constant 1000 nt/s rate, meaning that the 4.6 X
106 bp E. coli chromosome has a replication time, C of ~40 min.
The segregationof cellular components and formation of septum has a
fixed time of D = 20 min, after the completion of the corresponding
round of chromosome replication.
Cells with doubling times < C + D = 60 min must initiate chromosome
replication before the end of the preceding cell division cycle.
This results in multiforked chromosomes.
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Figure 30-30
Multifork
ed
chromosomes
in E. coli. In
cells that are
dividing every
35 min, the
fixed 60-min
interval
between the
initiation of
replication and
cell division
results in the
production of
multiforked
chromosomes.
How does DNA move to the new cell?
• Dam methyltransferase methylates GATC sequences
in E. coli.
• GATC occurs 11 times in oriC.
• Newly replicated GATC segments are hemimethylated
(new strands not methylated).
• Hemimethylated DNA associated with the cell
membrane.
• Interacts with SeqA protein for the sequestration of
DNA based on the oriC site.
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Figure 30-31 Electron micrograph of an intact and
supercoiled E. coli chromosome attached to two
fragments of the cell membrane.
DNA Damage and Repair
•
DNA is the instruction manual for the cell.
•
Changes in DNA base sequence are called mutations.
•
Most mutations are harmful, even lethal to cells.
•
Silent mutations are changes in DNA sequence that do
not affect the function of protein products.
•
Mutations can occur by spontaneous processes or
induced processes.
•
Final error rate in E. coli replication: ~1 x 1010 base pairs.
•
Actual error rate of base incorporation during E. coli
replication: 1 in 104-105 bases inserted.
•
Conclusion: repair systems correct most mismatched
bases.
DNA Damage and Repair
•
Mutations are usually bad, but may be responsible for
selective advantages, evolutionary processes.
•
Mutations can occur by spontaneous processes or
induced processes.
Two types of spontaneous mutation processes:
1. Mistakes in the incorporation of deoxyribonucleotides
(mismatched base pairs: A-C, G-T).
2. Base modifications caused by hydrolytic reactions - removal of
purine base ring by hydrolysis at the N-glycosidic bond.
Three types of replication errors:
1. Point mutation - substitution of one base pair for another. (Most
common, results from base tautomerism)
2. Insertion of one or more extra base pairs.
3. Deletion of one or more base pairs.
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Figure 30-51 Types and sites of chemical
damage to which DNA is normally
susceptible in vivo. Red, oxidation; blue,
hydrolysis; green, methylation.
Mutagenic damage to DNA caused by spontaneous
processes
Note conversion of cytosine to uracil in (b).
Secondary or indirect damage to DNA caused by the
hydroxyl radical, OH.
The OH radical is
formed by
interaction of
The OH removes
a hydrogen atom
from DNA,
forming H2O and
a reactive DNA
radical.
This results in a
broken DNA
strand.
Some sources of ionizing radiation:
1.
2.
3.
4.
5.
6.
7.
8.
Cosmic rays
Medical X-rays
Nuclear weapons testing (fallout)
Nuclear power plants
Airplane travel (high altitude)
Radon gas in poorly ventilated residential buildings
Radium mining tailings (waste rock - radon gas)
Forgotten radium processing sites
Chemical Mutagens.
•
Heterocyclic base analogs like these are incorporated
into replicating DNA and induce mutations by altering
base-pairing characteristics.
Intercalating agents as chemical mutagens
Flat , hydrophobic , typically
aromatic molecules that
insert between base pairs in
DNA.
Figure 11.17 Binding of intercalating agents to DNA,
which causes structural distortions
DNA Damage and Repair
•
Different responses to damage
•
Direct reversal of damage
•
Example: photolyase corrects thymine dimers
•
Cyclobutylthymine dimers form under UV radiation.
•
These pyrimidine dimers distort the DNA base pair
structure.
•
Photolyases found in prokaryotes and eukaryotes
•
Bind to pyrimidine dimers and have a noncovalently
bound chromophore (MTHF) that abs. 300-500nm light
and transfers energy to FADH which cleaves the dimer.
•
Mechanism through base flipping (distortion of the
double helix).
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Figure 30-52 The cyclobutylthymine dimer
that forms on UV irradiation of two adjacent
thymine residues on a DNA strand.
Photolyases repair pyrimidine dimers
•
•
•
•
•
•
Pyrimidine dimers distort DNA structure so that it cannot be
transcribed or replicated.
A single thymine dimer is enough to kill E. coli if unrepaired.
Repaired by photolyases-bind in the dark, active in light
Use a noncovalenly bound chromophore (N5,N10methylenyltetrahydrofolate (MTHF) or 5-deazaflavin)
absorbs 300 to 500 nm light and transfers energy to FADH- which
transfers energy to break dimer.
Resulting pyrimidine anion reduces FADH• and repaired DNA is
released.
Figure 30-53 X-Ray structure of E. coli
DNA photolyase showing its putative DNA
binding surface.
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Pyrimidine
binding site
DNA Damage and Repair
•
Alkyltransferases dealkylate alkylated nucleotides
•
Exposure of DNA to N-methyl-N’-nitrosoguanidine
(MNNG) will alkylate purines.
Reactive methylating agents shown here can convert
guanine (pairs with C) to O6-methylguanine, which pairs
with thymine.
Figure 30-54aThe structure of E. coli Ada protein. (a) The X-ray
structure of Ada’s 178-residue C-terminal segment, which
contains its O6-alkylguanine–DNA alkyltransferase function.
Alkyltransferases
dealkylate alkylated
nucleotides
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Transfers the alkyl group
to an active Cys residue
at 321.
Must undergo
conformational change in
order to effect methyl
transfer.
N terminus repairs
methyl phosphotriesters
by binding to Cys 69
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Figure 30-54b
The structure of E. coli Ada protein.
(b) The NMR structure of Ada’s 92-residue, N-terminal
segment, which mediates its methyl phosphotriester
repair function.
Ada has two independent functions
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•
•
•
C-terminus repairs O6-alkylguanine DNA through
transfer of methyl group to Cys 321.
N-terminal segment repairs methyl phosphotriesters in
DNA by transferring methyl group to Cys 69
N-terminus has a Zn atom that stabilized the thiolate
form over thiol form (-S- vs -SH)
-S- can attack the methyl group on DNA
Excision Repair
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Two types
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(1) nucleotide excision repair (NER) repairs bulky DNA
lesions
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(2) base excision repair (BER) repairs a single base.
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NER in prokaryotes uses three subunits, eukaryotes 16
subunits.
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UvrA, UvrB, and UvrC cleave the damaged DNA strand at
the 7th and 3rd or 4th phosphodiester bonds from the
lesion’s 5’ and 3’ sides.
•
Excised 11 or 12 nt oligo is displaced by UvrD.
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Figure 30-55 The mechanism of nucleotide excision
repair (NER) of pyrimidine photodimers.
Excision Repair
•
(2) base excision repair (BER) repairs a single base.
•
Adenine and cytosine spontaneously deaminate to yield
hypoxanthine and uracil
•
S-Adenosylmethionine (SAM) occasionally methylates a
base to form 3-methyladenine and 7-mehtylguanine.
•
DNA glycosylaes cleave the glycosidic bond of altered
nucleotides leaving apurinic or apyrimidinic (AP) sites.
•
The deoxyribose residue is cleaved on one side by the AP
endonuclease, the other side by an exonuclease (DNA
polymerase) and gap is filled by polymerase and DNA
ligase.
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Figure 30-56 Action of DNA glycosylases. These
enzymes hydrolyze the glycosidic bond of their
corresponding altered base (red) to yield an AP site.
Mismatch Repair
• Repairs mispairings in DNA that have not been caught by
DNA polymerases and MMR can correct insertions or
deletions up to 4 nt.
•
Must distinguish the parental strand from the daughter
strand. In E. coli this is possible because the newly
replicated GATC palindromes remain hemi-methylated until
the Dam methyltransferase has had sufficient time to
methylate the daughter strand.
•
Requires three proteins: MutS, MutL and MutH
1. MutS binds to mismatched base pair or unpaired bases as a
homodimer.
2. The MutS-DNA complex binds to MutL homodimer
3. MutS-MutL translocates along the DNA forming a loop in the DNA.
4. Encountering a hemimethylated GATC palindrome, recruits MutH and
activates single strand endonuclease to make a nick on the 5’ side of
the unmethylated GATC.
Mismatch Repair
•
Repairs mispairings in DNA that have not been caught by
DNA polymerases and MMR can correct insertions or
deletions up to 4 nt.
•
Must distinguish the parental strand from the daughter
strand. In E. coli this is possible because the newly
replicated GATC palindromes remain hemi-methylated until
the Dam methyltransferase has had sufficient time to
methylate the daughter strand.
•
Requires three proteins: MutS, MutL and MutH
Mismatch Repair
1. MutS binds to mismatched base pair or unpaired bases as a
homodimer.
2. The MutS-DNA complex binds to MutL homodimer
3. MutS-MutL translocates along the DNA forming a loop in the DNA.
4. Encountering a hemimethylated GATC palindrome, recruits MutH and
activates single strand endonuclease to make a nick on the 5’ side of
the unmethylated GATC. May be on either side of the mismatch and
over 1000 bp away from it.
5. MutS-MutL recruits UvrD helicase, which in concert with an
exonuclease separates the strands and degrades the nick strand to
beyond the mismatch.
6. Gap is filled by Pol III and sealed by DNA ligase.
Eukaryotes more complex; 6 homologs of MutS and 5 of MutL. MutH is
exclusive to gram-negative bacteria.
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Figure 30-58 The
mechanism of
mismatch repair in
E. coli.
SOS response
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SOS response causes cells to stop dividing and repair
damaged DNA.
•
LexA and RecA mutants always have the SOS response
on.
•
When E. coli is exposed to agents that damage DNA, RecA
mediates proteolytic cleavage of LexA. This is induced by
RecA binding to ssDNA.
•
LexA is a repressor of 43 genes involved in DNA repair (all
proceeded by 20 nt sequence called the SOS box).
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Figure 30-59 Regulation of the SOS response in E. coli.