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DNA Synthesis
3'
3'5'
SSB
5'
Leading Strand
5'
DNA Pol I
3'DNA Ligase
Lagging Strand
HelicaseGyrase
3'
5'
DNA Pol III
Primase
RNA primers DNA templateNew DNA Telomere
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E. coli DNA Polymerase III
Processive DNA SynthesisThe bulk of DNA synthesis in E. coli is carried out by the DNA polymerase III holoenzyme.
• Extremely high processivity: once it combines with the DNA and starts polymerization, it does not come off until finished.
• Tremendous catalytic potential: up to 2000 nucleotides/sec.
• Low error rate (high fidelity) 1 error per 10,000,000 nucleotides
• Complex composition (10 types of subunits) and large size (900 kd)
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E. coli Pol III: an asymmetrical dimer
Polymerase Polymerase
Stryer Fig. 27.30
clamp loader
Sliding clamp
3'-5' exonuclease
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2 sliding clamp is important for processivity of Pol III
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DNA Synthesis
3'
3'5'
SSB
5'
Leading Strand
5'
DNA Pol I
3'DNA Ligase
Lagging Strand
HelicaseGyrase
3'
5'
DNA Pol III
Primase
RNA primers DNA templateNew DNA Telomere
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Stryer Fig. 27.33
Lagging strand loops to enable the simultaneous replication of both DNA strands by dimeric DNA Pol III
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DNA Ligase seals the nicks
OH P
O
-O O
O-
O P
O
O
O-DNA Ligase + (ATP or NAD+)
AMP + PPi
• Forms phosphodiester bonds between 3’ OH and 5’ phosphate• Requires double-stranded DNA• Activates 5’phosphate to nucleophilic attack by trans-esterification with activated AMP
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DNA Ligase -mechanism
1. E + ATP E-AMP + PPi
OH +DNA-3' P
O
AMP-O O
O-
5'-DNA O P
O
O
O-
DNA-3' 5'-DNA
+ AMP-OH
3.
2. E-AMP + P-5’-DNA P
O
AMP-O O
O-
5'-DNA
(+)H2NP
O
O(-)
O
OH
O
Ade
ENZYME
OH
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DNA Synthesis in bacteria: Take Home Message
1) DNA synthesis is carried out by DNA polymerases with high fidelity.
2) DNA synthesis is characterized by initiation, priming, and processive synthesis steps and proceeds in the 5’ 3’ direction.
3) Both strands are synthesized simultaneously by the multisubunit polymerase enzyme (Pol III). One strand is made continuously (leading strand), while the other one is made in fragments (lagging strand).
4) Pol I removes the RNA primers and fills the resulting gaps, and the nicks are sealed by DNA ligase
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Eukaryotic vs prokaryotic cells
Prokaryotes:
• no membrane-bound nucleus
• transcription and translation are coupled
Eukaryotes:
• DNA is located in membrane-bound nucleus
• Transcription and translation are separated in space and time
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DNA replication in eukaryotes
Similarities with E.coli replication
1. Polynucleotide chains are made in the 5’ 3’ direction2. Require a primer (RNA).3. Similarities with the E Coli DNA Pol active site and tertiary structure
Differences
1. Eukaryotic replication is much slower (only 100 nt/sec).2. Many replication origins.3. DNA is associated with histones.4. DNA Polymerases are more specialized, and their interactions
are more complex.4. Chromosomal DNA is linear -> requires special processing of
the ends.
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Eukaryotic DNA has many replication origins
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Cell Cycle
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Eukaryotic DNA polymerases
Size, kd
3’- exo
Function Notes
Pol
250 no chromosomal DNA replication
Inhibited by arabinosyl NTPs
Pol
39 DNA repair Inhibited by dideoxy NTPs
Pol 170 yes chromosomal DNA replication
Inhibited by arabinosyl NTPs
Pol
200 yes DNA replicationin mitochindria
Inhibited by dideoxy NTPs
Pol 260 yes DNA repair Inhibited by aphidocolin
Pol
lesion bypass
Pol
lesion bypass
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Analogy between bacterial and eukaryotic proteins involved in DNA replication
Bacteria Eukaryotes
SSB RPAPol I polymerase Pol Pol III polymerase Pol 2 subunit of Pol III PCNA3’ exonuclease of Pol I RnaseH + FEN1 subunit of Pol III RCF
RPA = Replication protein A PCNA = proliferating cell nuclear antigenFEN1 = flap endonuclease
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Lagging strand synthesis in eukaryotes
RPA
Pol/primase
5’
5’
(a)
(b)
5’
RNA primer
RCFPCNA
(c)
5’
Pol
(d)
(e)
Rnase H/FEN1
(f)
RPA=Replication protein A
10-30 nt
RCF = clamp loaderPCNA = sliding clamp
RnaseH = 5’-nucleaseFEN1 = flap endonuclease
ligase
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Telomerase preserves chromosomal ends
• The ends of the linear DNA strand cannot be replicated due to the lack of a primer • This would lead to shortening of DNA strands after replication
RNA primer
5‘… 3'3‘… 5'
• Solution: the chromosomal ends are extended by DNA telomeraseThis enzyme adds hundreds of tandem repeats of a hexanucleotide(AGGGTT in humans) to the parental strand:
5‘… 3'3‘… 5'
AGGGTTAGGGTTAGGGTT…
telomere
5‘… 3'3‘… 5'
AGGGTTAGGGTTAGGGTT…TCCCAATCCCAATCCCAA…
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RNA primer
Upstream Okazakifragment
Circular DNA does not have ends:
5‘… 3'3‘… 5'
Linear DNA:
RNA primer
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Telomerase is a reverse transcriptase that uses it own RNA as a template for elongation of the 3’ end of DNA
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Telomerase mechanism
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Telomerase mechanism - continued
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Telomeres form G-tetraplex structures
N
NH
N
N
O
NH2
N
NH
N
N
OH2N
N
HN
N
N
O
H2N
N
HN
N
N
ONH2
GG
G
G
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Telomerase inhibitors
1. Telomerase RNA as a target for antisense drugs
2. G-tetraplexes at chromosomal ends as a drug target.
Porphyrins, anthraquinones: stabilize G-tetraplex structure, inhibit telomerase activity.
Modified oligonucleotides that hybridize with telomerase RNA, preventing it from beingused as a template for telomere synthesis.
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Termination of Polymerization:The Key to Nucleoside Drugs
HOO
N3
N
NH
O
O
HO
NH
N
N
HN
NH2N
HO
NH
N
N
O
NH2N
O
AZT Ziagen Acyclovir
HOO
OH
OH
N
N
NH2
O
AraC
Antiviral Antitumor
Principle of action: 1) cellular uptake2) activation to 5’-triphosphate3) incorporation in DNA resulting in chaintermination
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Nucleoside inhibitors of reverse transcriptase
DNA RNA Proteins Cellular Action
transcription translation
DNA
rep
licat
ion
Notable exception: retroviruses
RNA DNA
Reverse transcription
Proteins Cellular Action
translation
RNA
Typical flow of genetic information:
RNA
transcription
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Reverse transcriptases (RT) are RNA-directed DNA PolymerasesUsed by RNA viruses (HIV-I , human immunoblastosis virus,Rous sarcoma virus)
1. Make RNA-DNA hybrid (use its own RNA as a primer)2. Make ss DNA by exoribonuclease (RNase H) activity 3. Make ds DNA incorporate in the host genome
RNA RNA:DNA hybrid
RT RT
ss DNA
RNAse H
RT
ds DNA
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HIV Life Cycle
1 = Entry in CD4+ lymphocytes
2 = Reverse transcription
3 = Integration
4 = Transcription
5 = Translation
6 = Viral Assembly
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Termination of Polymerization:Nucleoside Drugs
HOO
N3
N
NH
O
O
HO
N
N
N
HN
NH2N
HO
NH
N
N
O
NH2N
O
AZT Ziagen Acyclovir
HOO
OH
OH
N
N
NH2
O
AraC
Antiviral Antitumor
Principle of action: 1) cellular uptake2) activation to 5’-triphosphate3) competition with normal substrate and incorporation in DNA resulting in chaintermination
(zidovudine)
Other examples: dideoxycytidine, dideoxyinosine
(abacavir)
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Anti-HIV drug Ziagen was discovered at the U of M College of Pharmacy
HO
N
N
N
HN
NH2N
Ziagen (abacavir)
Robert Vince, ProfessorDepartment of Medicinal Chemistry
1998
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Nucleoside Drugs Must Be Converted to
Triphosphates to be Part of DNA and RNA
HOO
OH
OO
OH
PHO
HO
O
OO
OH
P
O
P
OHO
HOO
OH
Base Base
BaseO
O
OH
P
O
P
O
O
OHBase
OH
OP
OHO
HO
Kinase
Kinase
Kinase
Monophosphate
DiphosphateTriphosphate
ATP
ATP
ATP
• Compete with normal substrate for RT binding• Cause chain termination
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DNA Chain termination by Nucleoside Analogs
O
OH
OPO
O
O-
3'
Template Strand
Primer Strand
Base
OPOPOP-O
OOO
O- O- O-
Mg2+
5'
Base
ZiagenNo 3’OH!
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Mechanisms of selectivity
1. Activated drug is recognized and incorporated in DNA only by reverse transcriptase, not by cellular DNA polymerases (RNA viruses).
• viral polymerases usually have lower fidelity(no proofreading)
• Mammalian DNA polymerases are more accurate
2. The drug is phosphorylated by viral kinase, notby cellular kinases (e.g. AZT).
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Mechanisms of resistance and possible solutions:
1. The drug cannot enter cells or is pumped out rapidly.2. The drug is rapidly deaminated to inactive form or normal substrate is
overproduced.3. The drug is no longer recognized by kinases and is not activated to triphosphate form.
Possible solution:Use activated phosphate form of nucleosides (Viread)
4. Activated drug is not incorporated in DNA by mutant reversetranscriptase (usually HIV RT mutations at codons 184,65,69, 74, and 115).
Possible solution: Use a mixture of several RT inhibitors (e.g. zidovudine (AZT) +
lamivudine (3TC) = Combivir®) or a mixture of different mechanisms of action (e.g. non-nucleoside RT inhibitors, protease inhibitors).
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Nucleoside inhibitors of DNA polymerase as anticancer drugs
HOO
OH
OH
N
N
NH2
O
AraC (1--D-arabinofuranosylcytosine)
• used for treating acute myelocytic leukemia • activated to triphosphate form by cellular kinases• causes inhibition of DNA synthesis, repair, and DNA fragmentation• very toxic
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DNA Damage, Mutations, and Repair
See Stryer p. 768-773
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DNA Mutations
1. Substitution mutations: one base pair for another, e.g. T for G• the most common form of mutation
• transitions; purine to purine and pyrimidine to pyrimidine
• transversions; purine to pyrimidine or pyrimidine to purine
2. Frameshift mutations
• Deletion of one or more base pairs
• Insertion of one or more base pairs
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Rare imino tautomer of A
N N
NH2
O
HN
NN
N
NH
C
• Very low rate of misincorporation (1 per 108 - 1 per 1010)• Errors can occur due to the presence of minor tautomers
of nucleobases.
Spontaneous mutations due to DNA polymerase errors
N
N
N
N
H2N
NH
N
O
O
AT
H3C
Normal base pairing Mispairing
10-4
amino
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A(imino)T
AT
A(imino)C
AT
GC
Final result: A G transition (same as T C in the other strand)
Consider misincorporation due to a rare tautomer of A
AT
1st
replication
5’3’
Normal replication
2nd replication
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Induced mutations result from DNA damage
Sources of DNA damage: endogenous
1. Deamination2. Depurination: 2,000 - 10,000 lesions/cell/day3. Oxidative stress: 10,000 lesions/cell/day
Sources of DNA damage: environmental
1. Alkylating agents2. X-ray 3. Dietary carcinogens4. UV light 5. Smoking
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N
NH
NN
O
NH2
N
N
NH2
O
G C
o
h
h
HN
NH
O
ON
N
NN
OR
NH2
TO6-AlkG
n
h
G A
GC
GT
AT
Normal base pairing in DNA and an example of mispairing via chemically modified nucleobase
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DNA oxidation
H3CNH
N
O
O
H3CNH
N
O
O
HO
HO
thymine glycol
NNH
NN
O
NH2
HN
NH
NN
O
NH2
O
8-oxo-G
Reactive oxygen species: HO•, H2O2, 1O2, LOO•
•10,000 oxidative lesions/cell/day in humans
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N N
NN
NH2
NNH
NN
O
NH2
N
N
NH2
O
N NH
NN
O
NNH
NH
N
O
O
NH
N
O
O
Hypoxanthine
Xanthine
Uracil
NNH
NN
O
N
N
NH
O
H
A G
Deamination
N N
NN
NH2
HO
N NH
NN
NH2HO
N NH
NN
O
Mechanism:
H2O
- NH3
A
G
C
C
Rates increased by the presence of NO (nitric oxide)
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Depurination to abasic sites
N NH
NN
O
NH2O
O
O
OHOO
O
Abasic site (AP site)
H2O
N NH
NNH
O
NH2
2,000 – 10,000/cell/day
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UV light-induced DNA Damage
NH
O
O
H3C
N
O
O
PO
O
O-
O
N
NH
O
O
CH3
NH
O
O
H3C
N
O
O
PO
O
O-
O
N
NH
O
O
CH3
…CC… Pyrimidine dimer
Easily bypassed by Pol (eta) in an error-free manner
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Deletions and insertions can be caused by intercalating agents
Stryer Fig. 27.44
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Metabolic activation of carcinogens
Stryer Fig. 27.45
N7-guanine adducts
G T transversions
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carcinogen or drug (X)
X
X
mutations
replication
**intact DNA
Chemical modifications of DNA in mutagenesis and anticancer therapy
metabolic activation
repair
DNA adducts
detoxification
reactive metabolite (X-)
DNA
excretion
cell death
Anticancer Cancer
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Importance of DNA Repair
• DNA is the only biological macromolecule
that is repaired. All others are replaced.
• More than 100 genes are required for DNA repair, even in organisms with very small genomes.
• Cancer is a consequence of inadequate DNA repair.
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DNA Repair Types
• Direct repair– Alkylguanine transferase– Photolyase
• Excision repair– Base excision repair– Nucleotide excision repair– Mismatch repair
• Recombination repair
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Direct repair
• DNA photolyase (E. Coli)
NH
O
O
H3C
N
O
O
PO
O
O-
O
N
NH
O
O
CH3
NH
O
O
H3C
N
O
O
PO
O
O-
O
N
NH
O
O
CH3
5'
3'
5'
3'
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N N
NN
O
NH2
CH3
O6-methylguanine
AGT-CH2-SH
N NH
NN
O
NH2
AGT-CH2-S CH3
Directly repaires O6-alkylguanines (e.g. O6-Me-dG, O6-Bz-dG)
In a stoichiometric reaction, the O6 alkyl group is transferred to a Cys residue in the active site. The protein is inactivated and degraded.
O6-alkylguanine DNA alkyltransferase (AGT)
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AGT protein is highly conserved
helix-turn-helix motif
hydrophobic side-chains form alkyl-binding pocket
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Excision Repair
Takes advantage of the double-stranded (double information) nature of the DNA molecule.
Four major steps:
1. Recognize damage.
2. Remove damage by excising part of one DNA strand.
3. The resulting gap is filled using the intact strand as the template.
4. Ligate the nick.
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Antiparallel DNA Strands contain the same genetic information
A ::
G :::
T ::
T
C
A
3'
3' 5'
5'
A ::
G
T ::
T
A
3'
3' 5'
5'
A ::
G :::
T ::
T
C
A
3'
3' 5'
5'
Original DNA duplex DNA duplex with one of the nucleotidesremoved
Repaired DNA duplex
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Excision Repair
Takes advantage of the double-stranded (double information) nature of the DNA molecule.
Four major steps:
1. Recognize damage.
2. Remove damage by excising part of one DNA strand.
3. The resulting gap is filled using the intact strand as the template.
4. Ligate the nick.
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Base excision repair (BER)
• Used for repair of small damaged bases in DNA (AP sites, methylated bases, oxidized bases…)
• Human BER gene hogg1 is frequently deleted in lung cancer
HN
NH
NN
O
NH2
O
8-oxo-G
OHOO
O
Abasic site (AP site)
NNH
NH
N
O
O
XanthineN N
NN
NH2
Me
N3-Me-Ade
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Base Excision Repair
O O
O
PO
O
O-
O
O
PO
O
O-
O
O
PO
O-
O-
Base1
OH
Base3
O O
O
PO
O
O-
O
O
PO
O
O-
O
O
PO
O-
O-
Base1
Base2
Base3
O O
OH
OH
PO
O
O-
O
O
PO
O-
O-
Base1
Base3
O O
O
PO
O
O-
O
O
PO
O
O-
O
O
PO
O-
O-
Base1
Base2
Base3
R
(a) (b) (c), (d)
a) modified base is excised by N-glycosylase b) the abasic site is cleaved by AP endonuclease/lyasec) the resulting gap is filled by Polymerase b d) DNA Ligase seals the nick
AP site
Base2-ppp
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BER enzyme AlkA complex with DNA
Stryer Fig. 27.48
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N
N
NH2
O
NH
N
O
O
Uracil
Uracil DNA glycosylase removes deaminated C
BERC
Not normally present in DNA
N
N
NH2
O
H3CNH
N
O
O
Thymine (T)Cytosine (C)
H3C
However, deamination of 5-Me-C produces thymine:
BER
Net result: G:T base pair
Normal DNA base
No Me group
Cytosine
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Nucleotide Excision Repair
• Corrects any damage that both distorts the DNA molecule and
alters the chemistry of the DNA molecule (pyrimidine dimers,
benzo[a]pyrene-dG adducts, cisplatin-DNA cross-links).
NH
O
O
H3C
N
O
O
PO
O
O-
O
N
NH
O
O
CH3
5'
3'
NH
NH
NN
NO
HO
HOOH
HOO
OH
• Xeroderma pigmentosum is a genetic disorder resulting in defective NER
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Nucleotide excision repair (NER)
exinuclease
Pol /
Mammalian Enzyme
DNA ligase
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Mismatch Repair Enzymes
Nucleotide mismatches can be corrected after DNA synthesis!
Repair of nucleotide mismatches:
1. Recognize parental DNA strand (correct base) and daughter strand (incorrect base)
Parental strand is methylated:
2. Replace a portion of the strand containing erroneous nucleotide (between the mismatch and a nearby methylated site –up to 1000 nt)
N
N
NH2
O
H3CN N
NN
HNMe
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Mismatch Repair in E. coli
Stryer Fig. 27.51
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Recombination repair
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DNA Synthesis in bacteria: Take Home Message
1) DNA synthesis is carried out by DNA polymerases with high fidelity.
2) DNA synthesis is characterized by initiation, priming, and processive synthesis steps and proceeds in the 5’ 3’ direction.
3) Both strands are synthesized simultaneously by the multisubunit polymerase enzyme (Pol III). One strand is made continuously (leading strand), while the other one is made in fragments (lagging strand).
4) Pol I removes the RNA primers and fills the resulting gaps, and the nicks are sealed by DNA ligase
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Genetic diseases associated with defective DNA repair
Xeroderma Pigmentosum NER
Hereditary nonpolyposis MMRcolorectal cancer
Cockrayne’s syndrome NER
Falconi’s anemia DNA ligase
Bloom’s syndrome BER, ligase
Lung cancer (?) BER