Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

250 Chapter 5: DNA Replication, Repair, and Recombination
unit (total molecular mass >10 6 daltons), enabling DNA to be synthesized on both
sides of the replication fork in a coordinated and efficient manner.
On the lagging strand, the DNA replication machine leaves behind a series of
unsealed Okazaki fragments, which still contain the RNA that primed their synthesis
at their 5ʹ ends. As discussed earlier, this RNA is removed and the resulting
gap is filled in by DNA repair enzymes that operate behind the replication fork
(see Figure 5–11).
A Strand-Directed Mismatch Repair System Removes Replication
Errors That Escape from the Replication Machine
As stated previously, bacteria such as E. coli are capable of dividing once every 30
minutes, making it relatively easy to screen large populations to find a rare mutant
cell that is altered in a specific process. One interesting class of mutants consists of
those with alterations in so-called mutator genes, which greatly increase the rate
of spontaneous mutation. Not surprisingly, one such mutant makes a defective
form of the 3ʹ-to-5ʹ proofreading exonuclease that is a part of the DNA polymerase
enzyme (see Figures 5–8 and 5–9). The mutant DNA polymerase no longer proofreads
effectively, and many replication errors that would otherwise have been
removed accumulate in the DNA.
The study of other E. coli mutants exhibiting abnormally high mutation rates
has uncovered a proofreading system that removes replication errors made by the
polymerase that have been missed by the proofreading exonuclease. This stranddirected
mismatch repair system detects the potential for distortion in the DNA
helix from the misfit between noncomplementary base pairs.
If the proofreading system simply recognized a mismatch in newly replicated
DNA and randomly corrected one of the two mismatched nucleotides, it would
mistakenly “correct” the original template strand to match the error exactly half
the time, thereby failing to lower the overall error rate. To be effective, such a proofreading
system must be able to distinguish and remove the mismatched nucleotide
only on the newly synthesized strand, where the replication error occurred.
The strand-distinction mechanism used by the mismatch proofreading system
in E. coli depends on the methylation of selected A residues in the DNA. Methyl
groups are added to all A residues in the sequence GATC, but not until some
time after the A has been incorporated into a newly synthesized DNA chain. As
a result, the only GATC sequences that have not yet been methylated are in the
new strands just behind a replication fork. The recognition of these unmethylated
GATCs allows the new DNA strands to be transiently distinguished from old ones,
as required if their mismatches are to be selectively removed. The three-step process
involves recognition of a newly synthesized strand, excision of the portion
containing the mismatch, and resynthesis of the excised segment using the old
strand as a template. This strand-directed mismatch repair system reduces the
number of errors made during DNA replication by an additional factor of 100 to
1000 (see Table 5–1, p. 244).
A similar mismatch proofreading system functions in eukaryotic cells but uses
a different strategy to distinguish the new strand from the old (Figure 5–19). Newly
synthesized lagging-strand DNA transiently contains nicks (before they are sealed
by DNA ligase) and such nicks (also called single-strand breaks) provide the signal
that directs the mismatch proofreading system to the appropriate strand. This
strategy also requires that the newly synthesized DNA on the leading strand be
transiently nicked; how this occurs is uncertain.
The importance of mismatch proofreading in humans is seen in individuals
who inherit one defective copy of a mismatch repair gene (along with a functional
gene on the other copy of the chromosome). These people have a marked predisposition
for certain types of cancers. For example, in a type of colon cancer called
hereditary nonpolyposis colon cancer (HNPCC), spontaneous mutation of the one
functional gene produces a clone of somatic cells that, because they are deficient
in mismatch proofreading, accumulate mutations unusually rapidly. Most cancers
arise in cells that have accumulated multiple mutations (see pp. 1096–1097),