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chapter 24
DNA Replication, Repair, and Mutagenesis
hematopoietic stem cells that do have residual telom-
erase activity, telomere shortening is observed at the level
of granulocyte and mononuclear cell fractions. Acceler-
ated telomere shortening has been observed in cells from
patients with aplastic anemia, suggesting that abnormal
telomere shortening is associated with disease and aging.
A characteristic of malignant tumor cells is that they
can replicate indefinitely. The immortality of tumor cells
appears to result, at least in part, from enhanced levels of
telomerase that allow them to repair and elongate telom-
eres at the ends of DNA. This hypothesis is supported by
the observation that ectopic expression of the catalytic sub-
unit of telomerase (a product of the
hTERT
gene) enabled
human retinal pigmented epithelial cells and fibroblasts
to avoid senescence and to maintain their differentiated
state when grown
in vitro.
Current research is focused on
drugs that can promote or inhibit the action of telomerase
or telomere-associated regulatory proteins. It is hoped that
increasing telomerase activity in cells approaching senes-
cence will retard aging or that decreasing telomerase ac-
tivity in tumor cells will result in arrested tumor growth.
24.5 DNA Repair
DNA can be damaged by external agents and by replication
errors. Since maintenance of the correct base sequence
of DNA and of daughter DNA molecules is essential for
hereditary fidelity, repair systems have evolved that restore
the correct base sequence.
Mismatch Repair and Methylation of DNA
DNA polymerases occasionally catalyze incorporation of
an “incorrect” base that cannot form a hydrogen bond with
the template base in the parental strand; such errors usu-
ally are corrected by the editing function of these enzymes.
The editing process occasionally fails, so a second sys-
tem, called
mismatch repair,
exists for correcting the er-
rors that are not edited out. In mismatch repair, a pair of
non-hydrogen-bonded bases (e.g., G • • • T; Figure 24-12a)
within a helix are recognized as aberrant and a polynu-
cleotide segment of the daughter strand is excised, thereby
removing one member of the unmatched pair. The result-
ing gap is filled in by pol I, which presumably uses this
“second chance” to form correct base pairs; then the final
seal is made by DNA ligase.
If it is only to correct and not create errors, the mismatch
repair system must be capable of distinguishing the cor-
rect base in the parental strand from the incorrect base in
the daughter strand. Rare methylated bases (methyl-A and
methyl-C) provide the basis for this distinction. In
E. coli,
■ ■ - 3' Template strand
GAGTCGAATC
FIGURE 24-12
Mismatch repair, (a) Excision of a short segment of a newly synthesized
strand and repair synthesis, (b) Methylated bases in the template strand
direct the excision mechanism to the newly synthesized strand containing
the incorrect nucleotide. The regions in which methylation is complete are
black lines; the regions in which methylation may not be complete are
shown in color.
an enzyme,
DNA methylase,
methylates adenine in the
sequence GATC. Methylation occurs soon (but not imme-
diately) after the replication fork has synthesized such a se-
quence in the daughter strand. Thus, the nucleotides in the
daughter strand are usually not methylated near the fork,
whereas those in the parental strand are always completely
methylated (Figure 24-12b). The mismatch repair system
recognizes the degree of methylation of a strand and pref-
erentially excises nucleotides from the undermethylated
strand. The daughter strand is always the undermethylated
strand, so that parental information is retained.
Glycosylases
Occasionally, uracil or other incorrect bases may become
incorporated into new DNA strands. These bases are usu-
ally removed by a pathway that starts with the cleavage
of the N-glycosidic bond by an enzyme called a gly-
cosylase (Figure 24-13). Many glycosylases are known,
and each is base-specific (e.g., uracil N-glycosylase). This
enzyme cleaves the N-glycosidic bond and leaves the de-
oxyribose in the backbone. A second enzyme (AP endonu-
clease) makes a single cut, freeing one end of the deoxyri-
bose. (AP stands for apurinic acid, a polynucleotide from
which purines have been removed by hydrolysis of the
N-glycosidic bonds.) This step is followed by removal of
the deoxyribose and several adjacent nucleotides (proba-
bly by a second enzyme that acts at the other side of the
apurinic site), after which pol I fills the gap with correct
nucleotides. This sequence, endonuclease-enlargement-
polymerase, is an example of a general repair mechanism
called
excision repair
(see below).
