Title: DNA Replication in Prokaryotes and Eukaryotes
1DNA Replication in Prokaryotes and Eukaryotes
- Overall mechanism
- Roles of Polymerases other proteins
- More mechanism Initiation and Termination
- Mitochondrial DNA replication
2Few Questions
- Is DNA replication conservative,
semi-conservative or random dispersed? - Is DNA replication bidirectional?
- Does DNA replication start at the same location
or random location?
3Replicon - DNA replicated from a single origin
Eukaryotes have many replication origins.
4Three Steps in DNA Replication
- Initiation
- Elongation
- Termination
5How is a Repl. origin selected? Priming at the
oriC (Bacterial) Origin
Initiation
6Figure 21.3
7OriC consensus sequence
245 bp minimal region
8 ATP
Hu on the origin
Ready to bind primase!
9Order of events at OriC
1. Many copies of dnaA bind the four 9-mers DNA
wraps around dnaA forming Initial Complex. This
requires ATP and a protein Hu that is already
bound to the DNA.
2. This triggers opening of the 13-mers (Open
complex).
3. Two copies of dnaB (helicase) bind the
13-mers. This requires dnaC (which does not
remain with the Prepriming Complex) and ATP.
4. Primase binds to dnaB (helicase) and the DNA.
5. dnaBprimase complex moves along the template
3gt5 synthesizing RNA primers 5gt3 for Pol III
to extend.
10 11Enzymes Involved in Elongation
- 1. DNA-dependent DNA polymerases
- synthesize DNA from dNTPs
- require a template strand and a primer strand
with a 3-OH end - all synthesize from 5 to 3 (add nt to 3 end
only)
12Movie DNA polymerization
13Comparison of E.coli DNA Polymerases I and III
1 subunit 10 subunits
14Proofreading Activity
Insertion of the wrong nucleotide causes the DNA
polymerase to stall, and then the 3-to-5
exonuclease activity removes the mispaired A nt.
The polymerase then continues adding nts to the
primer.
15If DNA polymerases only synthesize 5 to 3, how
does the replication fork move directionally?
16- Lagging strand synthesized as small (100-1000
bp) fragments - Okazaki fragments . - Okazaki fragments begin as very short 6-15 nt
RNA primers synthesized by primase.
2. Primase - RNA polymerase that synthesizes
the RNA primers (11-12 nt that start with pppAG)
for both lagging and leading strand synthesis
17Lagging strand synthesis (continued)
Pol III extends the RNA primers until the 3 end
of an Okazaki fragment reaches the 5 end of a
downstream Okazaki fragment.
Then, Pol I degrades the RNA part with its 5-3
exonuclease activity, and replaces it with DNA.
Pol I is not highly processive, so stops before
going far.
18At this stage, Lagging strand is a series of DNA
fragments (without gaps).
Fragments stitched together covalently by DNA
Ligase. 3. DNA Ligase - joins the 5 phosphate
of one DNA molecule to the 3 OH of another,
using energy in the form of NAD (prokaryotes) or
ATP (eukaryotes). It prefers substrates that are
double-stranded, with only one strand needing
ligation, and lacking gaps.
19DNA Ligase Substrate Specificity
20Mechanism of Prokaryotic DNA Ligase- Ligase
binds NAD, cleaves it, leaving AMP attached to
it. Ligase-AMP binds and attaches to 5 end of a
DNA molecule (1) via the AMP. The DNA fragment
with the 3 OH end (2) reacts with the
phosphodiester, displacing the AMP-ligase.
Ligase
NMN
NAD
AMP
N
M
N
3'
P
Ligase
AMP
3'
AMP
(Eukaryotic DNA ligase uses ATP as AMP donor,
instead of NAD).
N
A
D
HO
AMP
P
5'
3'
21Movie - Bidirectional Replication Leading and
lagging strand synthesis
22Other proteins needed for DNA replication 4.
DNA Helicase (dnaB gene) hexameric protein,
unwinds DNA strands, uses ATP.
5. SSB single-strand DNA binding protein,
prevents strands from re-annealing and from
being degraded, stimulates DNA Pol III.
6. Gyrase a.k.a. Topoisomerase II, keeps DNA
ahead of fork from over winding (i.e., relieves
torsional strain).
Replisome - DNA and protein machinery at a
replication fork.
23DNA Helicase (dnaB gene) Assay
Fig. 20.17 in Weaver
24Figure 20.18
Helicase
Primase
Fig. 20.18 in Weaver
25Helicase the movie
26DNA Supercoiling
27Rubber Band Model of Supercoiling DNA
DNA Gyrase relaxes positive supercoils by
breaking and rejoining both DNA strands.
28A Replisome