4
DNA Replication,
Repair, and Recombination
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Fig. 1. Watson-Crick A•T and G•C base pairs.
Fig. 2. Unwinding DNA at the origin of replication and the formation of replication forks. DNA
replication begins at specific sites known as origins of replication. Origin binding proteins recognize
these sites and initiate unwinding of the DNA duplex so that replication proteins can access the
individual strands of DNA. Initially a small bubble is formed that is opened further by the activity of
a DNA helicase. Replication complexes assemble on both sides of the bubble and these replication
forks (circled) move away from the origin in both directions so that replication is bidirectional. At
each fork, two new copies of DNA are synthesized using the parental strands as a templates.
Fig. 3. Initiation of DNA replication at oriC in E. coli. DnaA protein binds to oriC to form a proteinDNA complex where the DNA is wrapped around several molecules of DnaA protein. DnaA binding induces unwinding of the DNA duplex at the A•T rich segments. DnaC protein binds the
ring-shaped hexameric DnaB helicase and assembles the helicase onto the origin. One helicase
complex is assembled at each fork of the replication bubble. After assembling the helicase, DnaC
is released.
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Fig. 4. Proteins at the E. coli replication fork.The dimeric polymerase complex is capable coordinated DNA synthesis on the leading and lagging strands. The leading strand polymerase synthesizes new DNA in the direction of fork movement and the lagging strand polymerase synthesizes
DNA in the opposite direction. The hexameric helicase (light blue) unwinds DNA ahead of the
polymerase and primase (red) makes RNA primers (red lines) on the lagging strand. Single-stranded
DNA that forms as the helix unwinds is coated with single-stranded binding protein to prevent
reannealing of strands and to remove secondary structure that may form within a single-strand.
Sliding clamps (green) are assembled on each primer on the lagging strand by the clamp loading
complex (yellow and dark blue).
Fig. 5. Reactions catalyzed by DNA polymerases. (A) 2'-Deoxyribonucleoside 5'-triphosphates are
used as substrates by DNA polymerases to extend a primer in template-directed reactions. The net
reaction is incorporation of 2'-deoxyribonucleoside monophosphates onto the 3' hydroxyl of a primer
with loss of pyrophosphate. (B) DNA polymerases can proofread newly incorporated nucleotides
and excise incorrect nucleotides. The excision reaction removes the last nucleoside monophosphate
that was incorporated.
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A
B
Fig. 6. Structures of sliding clamps from E. coli and humans. (A) The E. coli β sliding clamp is a head-to-tail dimer
of identical monomer subunits. (B) The human PCNA sliding clamp is similar in overall structure to the β clamp
but is composed of identical trimers. Each ring has a central hole that is large enough to encircle B-DNA.
Fig. 7. Methyl-directed mismatch repair in E. coli. MutS protein recognizes and binds mismatches
such as G•T in DNA and is joined by the MutL protein. MutL within the MutS-MutL-mismatched
DNA complex stimulates the endonuclease activity of MutH to cleave the unmethylated DNA strand
at the GATC sequence closest to the protein-mismatched DNA complex. The cut DNA strand is
unwound by the activity of MutU helicase and then degraded by an exonuclease until the mismatch
is removed. The missing segment of DNA is replaced by a DNA polymerase and the DNA strands are
joined together by the activity of a DNA ligase. The letter P indicates a 5' phosphate group.
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A
B
Fig. 8. Homologous recombination. (A) Recombination between sister chromatids during meosis
results in exchange of information to generate two new chromatids that are hybrids of the originals.
(B) Double-strand break model for homologous recombination. In this model, recombination is
initiated by forming a double-stranded break (step 1) in one of the homologous duplexes. The
broken DNA is then processed by partial degradation by an exonuclease to generate single-stranded
DNA on the 3' ends (step 2). One 3' single-stranded end invades the homologous duplex forming
a D-loop in the intact duplex (step 3). The invading 3' end is extended by a DNA polymerase
enlarging the D-loop which can then pair with the remaining 3' single-stranded end (step 4). As the
D-loop expands, it can displace the 5' end of the broken duplex which is then free to pair with
the intact duplex (step 5). Branch migration enlarges the regions of heteroduplex DNA by unzipping the regions that were originally paired and zipping them onto the homologous duplex (step 6).
Finally, the cross-over points or Holliday junctions are resolved by cleavage of the crossing strands
(step 7). Two different products, patched and spliced, are formed depending on which of the crossed
strands are cleaved.
Fig. 9. Structures of a thymine cyclobutane dimer and 06-methylguanine.
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Fig. 10. Repair of DNA by excision of the damage and resynthesis of DNA. (A) The base excision
repair pathway begins with the removal of a damaged base by a DNA glycosylase. In this scheme
undamaged DNA bases are indicated by black squares and the damaged base is indicated by a
light square. The C1'-N glycosylic bond between the base and the sugar is cleaved leaving a baseless sugar residue (AP site) in DNA. The DNA strand is cut 5' to the AP site creating a 3' hydroxyl
on one side of the cut and a 5’phosphate (“P”) on the other. Deoxyribophos-phodiesterase activity
is required to excise the sugar-phosphate residue to create a one nucleotide gap that can be filled
in by a DNA polymerase. Repair is complete when the strands are ligated by a DNA ligase. (B) The
nucleotide excision repair pathway removes a short segment of DNA containing a damaged base
(red starburst). The damaged base is recognized and bound by a protein complex. This protein
complex serves to direct the other proteins to the site of damage so that it can be repaired. A DNA
helicase separates the DNA strands on either side of the damaged nucleotide. Specific endonucleases recognize the forked single-stranded/double-stranded DNA junctions at these sites and
cleave the DNA at the junctions. The DNA strand is cleaved 3' to the damaged nucleotide followed
by cleavage on the 5' side. The gap created by excision of the damaged DNA segment is filled in by
a DNA polymerase and the two strands are joined by a DNA ligase.
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