30.1 DNA Replication is Semiconservative
30.2 The Enzymology of DNA Replication
30.3 General Features of DNA Replication
30.4 The Mechanism of DNA Replication
30.5 Eukaryotic DNA Replication
30.6 Reverse Transcriptase
30.7 DNA Repair
April 25, 1953
Watson and Crick: "It has not escaped our notice that the specific (base) pairing we have
postulated immediately suggests a possible copying mechanism for the genetic material."
The mechanism: Strand separation, followed by copying of each strand.
Each separated strand acts as a template for the synthesis of a new complementary
strand.
The Semiconservative Model
Matthew Meselson and Franklin Stahl showed that DNA replication results in new DNA
duplex molecules in which one strand is from the parent duplex and the other is completely
new
Study Figure 30.4 and understand the density profiles from ultracentrifugation experiments
Imagine and predict the density profiles that the conservative and dispersive models would
show
of DNA Replication
If Watson and Crick were right, then there should be an enzyme that makes DNA copies
from a DNA template
In 1957, Arthur Kornberg and colleagues demonstrated the existence of a DNA
polymerase - DNA polymerase I
Pol I needs all four deoxynucleotides, a template and a primer - a ss-DNA (with a free
3'-OH) that pairs with the template to form a short double-stranded region
Replication occurs 5' to 3'
Nucleotides are added at the 3'-end of the strand
Pol I catalyzes about 20 cycles of polymerization before the new strand dissociates from
template
20 cycles constitutes moderate "processivity"
Pol I from E. coli is 928 aa (109 kD) monomer
In addition to 5'-3' polymerase, it also has 3'-5' exonuclease and 5'-3' exonuclease activities
Why the exonuclease activity?
The 3'-5' exonuclease activity serves a proofreading function! It removes incorrectly
matched bases, so that the polymerase can try again
See Figures 30.8 and 30.9! Notice how the newly-formed strand oscillates between the
polymerase and 3'-exonuclease sites, adding a base and then checking it
Nicks and Klenows....
5'-exonuclease activity, working together with the polymerase, accomplishes "nick
translation"
Hans Klenow used either subtilisin or trypsin to cleave between residues 323 and 324,
separating 5'-exonuclease (on 1-323) and the other two activities (on 324-928, the so-called
"Klenow fragment")
Tom Steitz has determined the structure of the Klenow fragment - see Figure 30.8
The "real" polymerase in E. coli
At least 10 different subunits
"Core" enzyme has three subunits - a,e, and q
Alpha subunit is polymerase
Epsilon subunit is 3'-exonuclease
Theta function is unknown
The beta subunit dimer forms a ring around DNA
Enormous processivity - 5 million bases!
Mostly in E. coli, but many features are general
Replication is bidirectional
The double helix must be unwound - by helicases
Supercoiling must be compensated - by DNA gyrase
Replication is semidiscontinuous
Leading strand is formed continuously
Lagging strand is formed from Okazaki fragments - discovered by Tuneko and Reiji "O"
Read page 947 on chemistry of DNA synthesis
DNA Pol III uses an RNA primer
A special primase forms the required primer
DNA Pol I excises the primer
DNA ligase seals the "nicks" between Okazaki fragments (See Figure 30.15 for
mechanism)
See Figure 30.16 for a view of replication fork
in E. coli
The replisome consists of: DNA-unwinding proteins, the priming complex (primosome) and
two equivalents of DNA polymerase III holoenzyme
Initiation: DnaA protein binds to repeats in ori, initiating strand separation and DnaB, a
helicase delivered by DnaC, further unwinds. Primase then binds and constructs the RNA primer
Elongation and Termination
Elongation involves DnaB helicase unwinding, SSB binding to keep strands separated, and
DNA polymerase grinding away on both strands
Termination: the "ter" locus, rich in Gs and Ts, signals the end of replication. A Ter protein
is also involved. Ter protein is a contrahelicase and prevents unwinding
Topoisomerase II (DNA gyrase) relieves supercoiling that remains
Like E. coli, but more complex
Human cell: 6 billion base pairs of DNA to copy
Multiple origins of replication: 1 per 3- 300 kbp
Several known animal DNA polymerases - see Table 30.4
DNA polymerase alpha - four subunits, polymerase (processivity = 200) but no
3'-exonuclease
DNA polymerase beta - similar to alpha
DNA polymerase gamma - DNA-replicating enzyme of mitochondria
DNA polymerase delta has a 3'-exonuclease as well as proliferating cell nuclear antigen
(PCNA)
PCNA give delta unlimited processivity and is homologous with prokaryotic pol III
DNA polymerase epsilon - highly processive, but does not have a subunit like PCNA
RNA-Directed DNA Polymerase
1964: Howard Temin notices that DNA synthesis inhibitors prevent infection of cells in
culture by RNA tumor viruses. Temin predicts that DNA is an intermediate in RNA tumor
virus replication
1970: Temin and David Baltimore (separately) discover the RNA-directed DNA
polymerase - aka "reverse trascriptase"
Primer required, but a strange one - a tRNA molecule that the virus captures from the
host
RT trascribes the RNA template into a complementary DNA (cDNA) to form a
DNA:RNA hybrid
All RNA tumor viruses contain a reverse transcriptase
Three enzyme activities
RNA-directed DNA polymerase
RNase H activity - degrades RNA in the DNA:RNA hybrids
DNA-directed DNA polymerase - which makes a DNA duplex after RNase H activity
destroys the viral genome
HIV therapy: AZT (or 3'-azido-2',3'- dideoxythymidine) specifically inhibits RT
A fundamental difference from RNA, protein, lipid, etc.
All these others can be replaced, but DNA must be preserved
Cells require a means for repair of missing, altered or incorrect bases, bulges due to insertion
or deletion, UV-induced pyrimidine dimers, strand breaks or cross-links
Two principal mechanisms: mismatch repair and methods for reversing chemical damage
Mismatch repair systems scan DNA duplexes for mismatched bases, excise the
mispaired region and replace it
Methyl-directed pathway of E. coli is example
Since methylation occurs post-replication, repair proteins identify methylated strand as parent,
remove mismatched bases on other strand and replace them
Pyrimidine dimers can be repaired by photolyase
Excision repair: DNA glycosylase removes damaged base, creating an "AP site"
AP endonuclease cleaves backbone, exonuclease removes several residues and gap is
repaired by DNA polymerase and DNA ligase
Note the box on page 961, discussing the relevance of correlations between life span and
DNA repair activity