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show 1000-fold increases in spontaneous mutation
frequencies compared to wild type polymerase. 104 Rates
for excising mismatched base pairs are typically 100-fold
higher than for correctly matched base pairs, 105 indi-
cating that mispaired primer-templates are the preferred
substrate for exonuclease activity. The overall mecha-
nism for nucleotide excision involves a competition
between the enzyme's polymerase and exonuclease
active sites for the 3'-end of the primer strand. 105 After
formation of the mismatch, polymerization is stalled
due to misorientation of the 3'-hydroxyl group of the
primer-terminus that subsequently hinders the incorpo-
ration of the next correct nucleotide. Polymerase stalling
enhances the probability of excision by the exonuclease
activity due to shuttling of the mispaired primer away
from the polymerization domain. In addition, a mis-
matched base pair destabilizes the duplex DNA, and
thus enhances the binding of the partially melted 3'-
single-stranded primer into the exonuclease site. Once
the primer is placed in the exonuclease domain, the
terminal nucleotide is hydrolyzed in a metal-dependent
reaction. The corrected primer is then shuttled back to
the polymerase domain so that correct DNA synthesis
can be renewed without the need for polymerase disso-
ciation and rebinding.
The process of exonuclease proofreading can be
altered by the presence of damaged DNA templates. If
a high-fidelity polymerase does incorporate a nucleotide
opposite a DNA lesion, exonuclease proofreading can
remove the nucleotide. However, due to higher intrinsic
processivity as well as interactions with other replicative
proteins, these DNA polymerases have an increased
opportunity to re-insert a nucleotide opposite the lesion
rather than dissociate. This process of repetitive addi-
tion, excision, and addition of a dNTP is called idle turn-
over and provides a kinetic barrier that should prevent
misreplication of DNA lesions by DNA polymerase
that are highly processive and that contain exonuclease
proofreading capabilities. 106 In addition to inhibiting
elongation beyond DNA lesions, this activity allows
the DNA polymerase to remain “stalled” on DNA and
may act to coordinate replication with other biological
pathways including DNA repair, DNA recombination,
and/or the by-pass of certain DNA lesions. 107
can act as physical barriers that inhibit DNA synthesis
by hindering movement of the DNA polymerase. In
other cases, alterations in the hydrogen-bonding infor-
mation present on nucleobases by DNA damaging
agents can enhance the frequency of misincorporation
events to increase pro-mutagenic DNA synthesis. The
formed mismatched base pairs are good substrates for
DNA repair enzymes, and activation of these various
enzymes can lead to repair of the lesion or cause cell
death. One excellent example of this phenomenon is
the use of temozolomide as a monofunctional alkylating
agent. Simple alkylation of the O 6 position of guanine
changes the hydrogen-bonding capabilities of the natural
base and causes more misincorporation events to occur
more frequently ( Figure 5.7A ). 108 The resulting mispair
caused by the misincorporation of dTMP opposite O 6 -
methylguanine activates mismatch repair pathway that
can lead to subsequent apoptosis. 109 More details
regarding the use of temozolomide in chemotherapy is
provided in this topic (see Chapters 2 and 9).
Monofunctional alkylating agents such as temozolo-
mide and BCNU create base damage or covalent
adducts to DNA that directly alter the speed and effi-
ciency of DNA replication. 110 However, these types of
DNA lesions are also processed by a number of repair
pathways which ultimately require the activity of vari-
ous DNA polymerases to complete the repair process.
As described above, the mismatch repair (MMR)
pathway actually leads to an enhancement in cytotoxic
effects of these by enforcing a futile cycle of repair and
resynthesis of undamaged DNA strand. However, other
pathways such as base excision repair (BER) and nucle-
otide excision repair (NER) can participate in the
removal of these lesions.
Other chemotherapeutic agents such as etoposide,
doxorubucin and ionizing radiation cause double-strand
DNA breaks (DSBs) that, by an obvious lack of coding
information, directly inhibit DNA synthesis. 111 DSBs are
repaired by non-homologous end-joining (NHEJ) and
homologous recombination (HR), each of which use
distinct DNA polymerases during the repair process.
The third most commonly used class of DNA damaging
agents are bifunctional alkylating agents suchas cisplatin,
chlorambucil and cyclophosphamide. 112,113 These agents
create crosslinks and/or bulky adducts that again form
physical barriers that impede DNA synthesis. If left unre-
paired, these lesions ultimately stall DNA synthesis and
cause SSBs and DSBs to form that can be repaired by
NHEJ and/or HR. Other repair pathways including
NER and the Fanconi anemia repair pathway participate.
Based on this cursory overview, it is clear that DNA
polymerases play important primary and secondary
roles in the therapeutic response to DNA damaging
agents. The focus of the next section is to probe the roles
of different DNA polymerases used in the repair of
Effects of DNA Damaging Agents
on DNA Polymerization
Amajor emphasis in chemotherapy has been to inhibit
processive DNA polymerases using DNA damaging
agents or nucleoside analogs. Most DNA damaging
agents react with functional groups present on the four
nucleobases and cause significant alterations in the struc-
ture of DNA and/or the hydrogen-bonding potential of
the nucleobase. In many cases, the formed DNA lesions
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