INFORMATION
COPYING: DNA
REPLICATION
INFORMATION
METABOLISM
Requires a template
Replication
Transcription
Translation
Each process consists of 3 distinct
subprocesses: initiation, chain
elongation and termination
GENETIC TERMINOLOGY
Genome
Genetic map: linkage map or physical map
Phenotype/genotype
Allele
Marker
Copy numeber
Plasmid
Wild-type
Reversion/ suppression (second-site
reversion)
EARLY INSIGHTS INTO DNA
REPLICATION
Semiconservative nature of replication
Sequential nature of replication
Origin and direction of replication
Units of replication
- initiation of replication is controlled by a small
cluster of genetic elements
-replicon
- a single chromosome is not always a single
replicon
-DNA replication is strongly controlled at the
level of initiation
Quantitative Parameters of Replication
[Link] Human
DNA content, 3.9 x 106 109
nucleotide pairs/cell
Rate of replication fork 30 μm/min 3 μm/min
progression
DNA replication rate 850 nucleotides/sec per 60-90 nucleotides/sec
fork per fork
Number of replication 1 103 – 104
origin per cell
Hours required to 0.67 8
complete geneome
replication
Hours to complete one 0.33 24
cell division
OVERVIEW OF DNA
REPLICATION
How does the coordination between cell division
and DNA replication occur?
How are the replication origins recognized by
enzymes?
What is the energy source for duplex strand
unwinding?
How many proteins must function to carry out
process?
How are the activities of these proteins
coordinated?
Is there an additional fidelity-enhancing
mechanism involved?
DNA POLYMERASE
POLYMERASE I
- most abundunt
-has polymerase and nuclease activity
-3’ exonuclease serves as a
proofreading function
-5’ exonuclease excise primers from the
lagging strand and repair DNA
(damaged by radiation or chemicals)
DNA POLYMERASE
POLYMERASE II and III
-pol III has a major role of nucleotide
incorporation during elongation
-pol II participates in DNA repair
synthesis
GENERAL STRUCTURE AND
MECHANISM
DNA POLYMERASE III
HOLOENZYME
CLAMP and CLAMP
LOADER
Uracil-DNA N-Glycosylase
DNA polymerases readily accepts
deoxyuridine triphosphate as a substrate
Removes dUMP residues
Hydrolytically cleaves the glycosidic bond
between N-1 uracil and C-1 deoxyribose
yielding an apyrimidinic site
Apyrimidinic endonuclease recognizes this
site and cleaves the phosphodiester bond
on the 5’ side of the deoxyribose moiety
INITIATION OF DNA REPLICATION
What are the site-specific DNA-protein
interactions that trigger initiation?
How do proteins act after binding to
origin sequence?
How is the process controlled?
REQUIREMENTS FOR INITIATION
A nucleotide sequence that specifically
binds initiation proteins
A mechanism that generates a primer
terminus to which nucleotides can be
added by DNA polymerase
In general, these origins include
repeated sequences of either identical
or opposite polarity
REQUIREMENTS FOR INITIATION
2 ways to generate a primer terminus:
1. Nicking a strand of the parental duplex
to expose the 3’ hydroxyl terminus
2. Unwinding the parental duplex and
synthesizing an RNA primer to expose
a 3’ hydroxyl ribonucleotide terminus
INITIATION of E. coli DNA
REPLICATION
Starts at the oric
245 bp long
Contains 4 repeats of 9 bp sequence
that binds an initiation protein DnaA
Contains 3 repeats of 13 bp sequence
that is rich in A and T
Also contains binding sites for proteins
HU and IHF that facilitate DNA bending
MITOCHONDRIAL DNA
REPLICATION
MITOCHONDRIAL DNA
REPLICATION
REPLICATION OF LINEAR
GENOMES
Phage T4 and T7
Bacteriophage ϕ29
Eukaryotes
Phage T4 and
T7
Bacteriophage
ϕ29
Eukaryotic DNA
Reverse Transcription
