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

E.coli 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