36
2 Nucleic Acids and Nuclear Proteins
polymerase is always associated with an exonuclease. Whilst the a-polymerase incorporates less
than 100 nucleotides before being released from
the substrate, the 6-polymerase has an in vivo
processive capacity of at least 4500 nucleotides.
This is related to the effect of an accompanying
36-kDa protein that is only produced in the Sphase of the cell cycle and is known as proliferating cell nuclear antigen (PCNA) or cyclin. In the
absence of PCNAlcyclin, the 6-polymerase is
released from its substrate after only ten incorporation steps [407].
Many polymerase complexes are simultaneously active on the chromosomal DNA, with the
gap between them (the replicon length) varying
from 5 (in embryos) to 350 !lm (in spermatogenesis). All new nucleosomes are apparently taken
up on the same daughter strand. In the Protozoa
and in the lower eukaryotes there are always
multiple DNA polymerases available but these
are only partially comparable with those of the
Eumetazoa. They have, without exception,
higher molecular masses, and in some cases display exonuclease activity [66, 146].
The ~-polymerases responsible for DNA
repair have no exonuclease activity and only a
limited accuracy; the error rate is 1:1000. Their
structure is much simpler than that of the other
polymerases. The enzymes of man and the mouse
are 39-kDa monomeric polypeptides with 335
amino acids [478]. Like the a-polymerases, these
enzymes also appear to have changed only very
slowly during evolution. The ~-polymerases of
different mammals are very similar immunologically and in their tryptic peptide patterns; antisera
against the rat enzyme react with the corresponding bird and fish enzymes but not with those of
the trypanosomes [64, 429]. Because ofits much
larger, 1l0-kDa subunits, the Drosophila ~polymerase belongs to a completely different
category [375]. The y-polymerase is responsible
for replication of the mitochondrial DNA and, as
it represents only about 1 % of the total DNA
polymerase activity of the cell, is known less
accurately; in pure mitochondrial preparations,
only the y-polymerase is found. However, the
enzyme is also found in cell nuclei, where it is
apparently involved in virus replication and possibly in DNA recombination processes. ypolymerase is characterized by a high molecular
mass (150-300 kDa), NEM sensitivity and the
exclusive use of DNA primers. The Drosophila
enzyme is a heterodimer of 125- and 35-kDa subunits. The enzyme of the clawed frog, Xenopus
laevis, has a catalytic subunit of 140 kDa and is
associated, like the chicken enzyme, with a 3'-5'exonuclease [206, 472].
Only rather sketchy information is available
about the other enzymes and protein factors
associated with the DNA polymerases of animals
[59], enzymes such as those required for unwinding the DNA and stabilizing the single strands, for
the synthesis of RNA primers, and nucleases and
ligases. In fact, Drosophila is the only animal
from which not only all three DNA polymerases
(a-y) but also primase, ligase and both topoisomerases have been isolated. Ligases create phosphodiester bonds between the free 5' -phosphate
groups and the free 3' -hydroxyl groups of broken
DNA strands. The ligase in the unfertilized eggs
of Drosophila melanogaster has the highest activity; this enzyme consists of only one 80- to 83kDa polypeptide [354, 355]. The topoisomerases
catalyse the reversible interconversion of different DNA isoforms, e.g. supercoiling and relaxation, or the reversible chain-like linking of circular DNA (catenation and decatenation). The
enzymes play important, but incompletely understood, roles in replication and transcription.
There are two very different forms of topoisomerases [466]. The type I nicking-closing enzymes
break one strand of the double helix and pull the
other strand through the gap in such a way that
the energy of the phosphodiester bond is conserved, and no further energy supply is required
for the reannealing process. Such enzymes have
been characterized in detail in various mammals,
the chicken, Xenopus, Drosophila and Trypanosoma cruzi. The type II topoisomerases open and
close both strands simultaneously and require
ATP. These enzymes are also well described for
mammals, Xenopus and Drosophila [356,359,
362,466,487].
Spontaneous alterations in DNA constituents
(point mutations) are of frequent occurrence in
all organisms; in man, for example, about 5000
purine bases per day are released from their Nglycosidic bonds (depurination), and 100 cytosine
residues are deaminated to uracil. A system of
repair enzymes deals with these and other forms
of damage; examples include DNA glycosylases,
exonucleases, DNA polymerases and ligases.
However, little is known about these from a comparative biochemical point of view [248, 376].
RNA-dependent DNA polymerases (reverse
transcriptases) are required for the transposition
of retrovirus-like DNA elements. High activities
of a reverse transcriptase have been found in the
ciliate Paramecium teraurelia but the biological
significance of this is not clear [226].
2 Nucleic Acids and Nuclear Proteins
polymerase is always associated with an exonuclease. Whilst the a-polymerase incorporates less
than 100 nucleotides before being released from
the substrate, the 6-polymerase has an in vivo
processive capacity of at least 4500 nucleotides.
This is related to the effect of an accompanying
36-kDa protein that is only produced in the Sphase of the cell cycle and is known as proliferating cell nuclear antigen (PCNA) or cyclin. In the
absence of PCNAlcyclin, the 6-polymerase is
released from its substrate after only ten incorporation steps [407].
Many polymerase complexes are simultaneously active on the chromosomal DNA, with the
gap between them (the replicon length) varying
from 5 (in embryos) to 350 !lm (in spermatogenesis). All new nucleosomes are apparently taken
up on the same daughter strand. In the Protozoa
and in the lower eukaryotes there are always
multiple DNA polymerases available but these
are only partially comparable with those of the
Eumetazoa. They have, without exception,
higher molecular masses, and in some cases display exonuclease activity [66, 146].
The ~-polymerases responsible for DNA
repair have no exonuclease activity and only a
limited accuracy; the error rate is 1:1000. Their
structure is much simpler than that of the other
polymerases. The enzymes of man and the mouse
are 39-kDa monomeric polypeptides with 335
amino acids [478]. Like the a-polymerases, these
enzymes also appear to have changed only very
slowly during evolution. The ~-polymerases of
different mammals are very similar immunologically and in their tryptic peptide patterns; antisera
against the rat enzyme react with the corresponding bird and fish enzymes but not with those of
the trypanosomes [64, 429]. Because ofits much
larger, 1l0-kDa subunits, the Drosophila ~polymerase belongs to a completely different
category [375]. The y-polymerase is responsible
for replication of the mitochondrial DNA and, as
it represents only about 1 % of the total DNA
polymerase activity of the cell, is known less
accurately; in pure mitochondrial preparations,
only the y-polymerase is found. However, the
enzyme is also found in cell nuclei, where it is
apparently involved in virus replication and possibly in DNA recombination processes. ypolymerase is characterized by a high molecular
mass (150-300 kDa), NEM sensitivity and the
exclusive use of DNA primers. The Drosophila
enzyme is a heterodimer of 125- and 35-kDa subunits. The enzyme of the clawed frog, Xenopus
laevis, has a catalytic subunit of 140 kDa and is
associated, like the chicken enzyme, with a 3'-5'exonuclease [206, 472].
Only rather sketchy information is available
about the other enzymes and protein factors
associated with the DNA polymerases of animals
[59], enzymes such as those required for unwinding the DNA and stabilizing the single strands, for
the synthesis of RNA primers, and nucleases and
ligases. In fact, Drosophila is the only animal
from which not only all three DNA polymerases
(a-y) but also primase, ligase and both topoisomerases have been isolated. Ligases create phosphodiester bonds between the free 5' -phosphate
groups and the free 3' -hydroxyl groups of broken
DNA strands. The ligase in the unfertilized eggs
of Drosophila melanogaster has the highest activity; this enzyme consists of only one 80- to 83kDa polypeptide [354, 355]. The topoisomerases
catalyse the reversible interconversion of different DNA isoforms, e.g. supercoiling and relaxation, or the reversible chain-like linking of circular DNA (catenation and decatenation). The
enzymes play important, but incompletely understood, roles in replication and transcription.
There are two very different forms of topoisomerases [466]. The type I nicking-closing enzymes
break one strand of the double helix and pull the
other strand through the gap in such a way that
the energy of the phosphodiester bond is conserved, and no further energy supply is required
for the reannealing process. Such enzymes have
been characterized in detail in various mammals,
the chicken, Xenopus, Drosophila and Trypanosoma cruzi. The type II topoisomerases open and
close both strands simultaneously and require
ATP. These enzymes are also well described for
mammals, Xenopus and Drosophila [356,359,
362,466,487].
Spontaneous alterations in DNA constituents
(point mutations) are of frequent occurrence in
all organisms; in man, for example, about 5000
purine bases per day are released from their Nglycosidic bonds (depurination), and 100 cytosine
residues are deaminated to uracil. A system of
repair enzymes deals with these and other forms
of damage; examples include DNA glycosylases,
exonucleases, DNA polymerases and ligases.
However, little is known about these from a comparative biochemical point of view [248, 376].
RNA-dependent DNA polymerases (reverse
transcriptases) are required for the transposition
of retrovirus-like DNA elements. High activities
of a reverse transcriptase have been found in the
ciliate Paramecium teraurelia but the biological
significance of this is not clear [226].