Page 127 - Academic Press Encyclopedia of Physical Science and Technology 3rd BioChemistry

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Encyclopedia of Physical Science and Technology en010k-502 July 16, 2001 16:56

Nucleic Acid Synthesis 865

eukaryotes are not only much larger and linear, but also ulator proteins, such as geminin, which blocks licensing,
contain multiple ori sequences for DNA replication and is also regulated by some cell cycle-dependent feedback
thus multiple replicons. Thousands of replicons are simul- mechanisms.
taneously fired in mammalian genomes, as is needed to
complete replication of the genome in a few hours. Mam-
G. Fidelity of DNA Replication
malian genomes are three orders of magnitudes larger than
the E. coli genome for which one round of replication re- The maintenance of genomic integrity in the form of the
◦
quires about 40 min at 37 C. Replication of a mammalian organism-specific nucleotide sequence of the genome is
genome, initiated at a single ori, would thus take more than essential for preservation of the species during propaga-
1 week with the same rate of synthesis. In fact, it would tion. This requires an extremely high fidelity of DNA
be even longer because the rate of DNA chain elongation replication. Errors in RNA synthesis may be tolerated at
is slower in eukaryotes than in E. coli, possibly because a significantly higher level because RNAs have a limited
of the increased complexity of eukaryotic chromatin. half-life, even in nondividing cells, and are redundant. In
As mentioned earlier, DNA replication in eukaryotes contrast, any error in DNA sequence is perpetuated in the
occurs only during the S phase, which can last for sev- future, as there is only one or two copies of the genome per
eral hours but whose duration varies with the organism, cell under most circumstances. Obviously, all organisms
the cell type, and also the developmental stage. For ex- have a finite rate of mutation, which may be necessary
ample, in a rapidly growing early embryo of the fruitfly for evolution. Genetic errors are one likely cause of such
D. melanogaster, cellular multiplication with duplication mutations. Inactivation of a vital protein function by muta-
of the complete genome occurs in less than 15 min. The tion of its coding sequence will cause cell death. However,
details of temporal regulation of firing of different repli- mutations that affect nonessential functions could be tol-
cons are not known. However, euchromatin regions are erated. Some of these mutations can still lead to change in
replicated earlier than the heterochromatin regions. thephenotype,whichinextremecasescancausepatholog-
Thedetailsofinitiationofreplicationatindividualrepli- ical effects. In other cases, these may be responsible for
cons have not been elucidated in eukaryotes. Some ori susceptibility to diseases. In many cases, however, such
sequences of the yeast genome, known as autonomous mutations appear to be innocuous and are defined as an
replication sequences (ARS), have been determined. Al- allelic polymorphism. The mammalian genome appears
though such sequences in the mammalian genomes have to have polymorphism in one out of several hundred base
not been isolated, the ori regions of certain genes which pairs. Such mutations obviously arose during the evolution
could be selectively amplified have been localized by two- and subsequent species propagation.
dimensional electrophoretic separation. Nevertheless, a The error rate in replication of mammalian genome
significant amount of information has been gathered re- is about 10 −6 to 10 −7 per incorporated deoxynucleotide.
garding regulation of DNA replication at the global level. The catalytic units of the replication machinery, namely,
DNA polymerases, have a significantly higher error rate
of the order of 10 −4 to 10 −5 per deoxynucleotide. In fact,
F. Licensing of Eukaryotic Genome Replication
some DNA polymerases, notably the reverse transcrip-
Unlike in bacteria and plasmids, DNA replication in eu- tases of retroviruses, including HIV, the etiologic agent
karyotic cells is extremely precise, and replication initia- for AIDS, are highly error prone and incorporate a wrong
3
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tion occurs only once in each cell cycle to ensure genomic nucleotide for every 10 –10 nucleotides. These mistakes
stability. “Licensing” is the process of making the chro- result in a high frequency of mutation in the viral pro-
matin competent for DNA replication in which a collec- tein, which helps the virus escape from immunosurveil-
tion of proteins called origin recognition complex (ORC) lance. The overall fidelity of DNA replication is signifi-
bind to the ori sequences. This binding is necessary for cantly enhanced by several additional means. The editing
other proteins required for the onset of the S phase to bind or proof-reading function of the replication machinery is
to DNA. ORC is present throughout the cell cycle. How- a3 → 5 exonuclease (which is either an intrinsic activ-

ever, other proteins required for replication initiation and ity of the core DNA polymerase or is present in another
chain elongation are loaded in a stepwise fashion. The subunit protein of the replication complex) which tests
onset of the S phase may be controlled by a minichromo- for base pair mismatch during DNA replication and re-
some maintenance (MCM) complex of proteins which moves the misincorporated base. Such an editing function
licenses DNA for replication, presumably by making it is also present during RNA synthesis. In addition, after
accessible to the DNA synthesis machinery. Several pro- replication is completed, the nascent duplex is scanned
tein factors are involved in the loading process, which is for the presence of mispaired bases. Once such mispairs
regulated both positively and negatively. The level of reg- are marked by mismatch recognition proteins, a complex