Outline for Replication
I. General Features of Replication
A. Semi-Conservative
B. Starts at Origin
C. Bidirectional
D. Semi-Discontinuous
II. Proteins and Enzymes of Replication
III. Detailed Examination of the Mechanism of Replication
A. Initiation
B. Priming
C. Elongation
D. Proofreading and Termination
DNA replication with
two forks
DNA replication Fork
The polarity of DNA synthesis creates an asymmetry
between the leading strand and the lagging strand
at the replication fork
Protein complexes of the replication fork:
DNA primase
DNA Helicase
ssDNA binding protein
DNA Ligase
DNA Topoisomerase
DNA polymerase
RNaseH
Sliding Clamp
Clamp Loader
Replication caught in Action
Topoisomerase
II. Identifying Proteins and Enzymes involved in Replication
A. Combine Genetics and Biochemistry
1. Genetic Approach: Obtain Mutants that are defective in Replication
a. Such mutations are Lethal!
b. Conditional lethal
c. Temperature sensitive (ts) lethal
2. Method:
a. Mutagenize cells
b. Plate the cells on agar plates and grow at 30 oC
c. Replica plate and grow at 37oC
2 classes of DNA replication mutants identified by Francis Jacob et al
1. Quick-stop mutants: major class
Stop replication immediately upon increase in temperature.
 Defect in elongation enzymes, enzyme that makes precursors
2. Slow-stop mutants: small class
Complete current round of replication but cannot start next one
> Replication initiation defect
In vitro complementation:
A technique used to identify the mutants in DNA Replication (dna mutants)
Make extract from dna mutant grown at restrictive temperature
and complement with wt components to identify dna defect
ts mutant WT (fraction X)
DNA
DNA synthesis
37C
Purify DnaX component
from fraction X
Priming of DNA replication
Examples:
Replication
Rolling circle
Replication
Protein A nicks
X174 viral strand
at ori
DNA primase
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Primase is a special RNA polymerase that makes RNA primer on a
ssDNA template
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Unlike DNA Polymerase, primase can start synthesis de novo
Makes an RNA primer of 4-12 nucleotides
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Different from other RNA polymerase: no sequence specificity
How is it regulated? Activated only when bound by DNA replication
proteins like helicases
PRIMOSOME: complex formed between primase and helicase
DnaG is primase in E. coli; it associates with RF proteins at oriC
Primase is not rifampicin sensitive while RNA pol is sensitive
EXCEPTION: M13 phage uses bacterial RNA pol for primer synthesis
Yeast: PRI1 and PRI2
Humans: primase (PRIM1 , 2)
Primase also acts as a halting mechanism to prevent the leading strand from
outpacing the lagging strand by halting the progression of the replication
fork.
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LEADING STRAND synthesis needs only 1 RNA primer
LAGGING STRAND requires multiple (100-1000) RNA primers
DNA Primer synthesis
On Lagging strand
Lagging strand
DNA Replication at the leading
strand
DNA Helicase
(essential for cell survival)
• separates or unwinds DNA strands in a
double-helix using ATP hydrolysis (1
ATP/bp unwound); thus an ATPase
•Thus creates ssDNA templates for
replication
• Functions as a hexamer
•Forms a ring and translocates along
ssDNA
•Unidirectional (5’ to 3’)
•Acts processively: helicase remains
bound to substrate for long time
•DnaB is helicase in E. coli (12
helicases in all): identified as quick stop
mutant
•Yeast and humans: Mcm complex (2-7)
Structure of a DNA Helicase
DNA replication fork
and helicase to scale.
(C) Detailed structure of the bacteriophage T7
replicative helicase,
DNA Helicase Assay
SSB: Single Strand DNA-binding Proteins, also called helix
destabilizing proteins
SSB (essential gene)
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Binds and maintains templates in single-stranded state
Binds ssDNA as tetramer
Sequence-independent binding
Co-operative binding: binding of 1 SSB to DNA promotes
binding of another SSB by inter-SSB interaction; ensures
rapid coating of ssDNA
SSB binding makes ssDNA into an extended conformation
which inhibits formation of hydrogen bonds
Types:
SSB in E. coli
Yeast and humans: RPA (Replication Protein A);
heterotrimeric protein
Structure of human SSB Proteins bound to DNA
DNA
Topoisomerases
DNA unwinding by helicase
creates positive supercoils ahead of
Replication forks
The supercoiling ahead
of the fork needs to be
relieved or tension would
build up (like coiling as
spring) and block fork
progression.
Supercoiling is relieved by the action of Topoisomerases.
Type I topoisomerases: no ATP required
Make nicks in one DNA strands
Can relieve supercoiling
Type II topoisomersases: ATP-dependent reaction
Make nicks in both DNA strands (double strand break)
Can relieve supercoiling and untangle linked DNA helices
Both types of enzyme form covalent intermediates with the DNA
E. coli: Gyrase (TopoII-like enzyme), TopoI
Eukaryotes: both I and II function in replication
DNA topoisomerase I
DNA topoisomerase II
RNAseH: A special DNA
repair enzyme that recognizes an
RNA strand in an RNA/DNA
hybrid and degrades it; thus
removes RNA primers;
this leaves gaps that are filled in by
DNA polymerase
Nicks in phosphate backbone are
sealed by DNA ligase.
RNAseH from E.coli
DNA Ligase:
Uses ATP energy to make phosphodiester bond
2-step reaction:
1. ligase-AMP complex forms
2. AMP complex attaches to 5’phosphate at the nick
then a phosphodiester bond is formed with the 3'-OH
terminus of the nick, thus releasing AMP and enzyme.
Mammalian replication Fork
(eucaryote, DNA polymerase (primase) a synthesize RNA/DNA,
DNA polymerase delta is the real polymerase)