The Complete "Replication Apparatus Is Complex When Watson and Crick worked out the double

helix structure of DNA, they immediately recognized that the complementary nature of the two
strands prcvided a simple basis for the faithful duplication of genetic material. Meselson and Stahl's
demonstration of the semiconservative replication of the E. coli chromo- some solidified the concept
that the two strands of the double helix unwind and serve as templates for the synthesis of
complementary strands. Thus, a parental double helix directs the synthesis of two identical progeny
double helices. Kornberg's isolation of an enzyme, DNA polymerase I, capable of synthesizing DNA in
vitro appeared to provide the final link in what was thought to be an elegantly simple mechanism for
the replication of the genetic materlal-but such was not the case. Twenty years later, scientists are
still trying to work out the details of the mechanism of DNA replication DNA replication is complex. It
is carried out by a multienzyme complex, often called the replicatiorn apparatus or the replisome. In
eukaryotes, the com- ponents of the replication machirery are just begin-ning to be identified. Even
in prokaryotes, DNA reproduction requires many different proteins, and the details of the function
proteins in DNA replication are still being investigated today. For example DNA replication in E. coli
requires at least a dozen different gene-products. Many of these gene-products have been purified
and their roles in DNA replication were studied in vitro. Figure 5.30 shows the E. coli proteins in DNA
replication; First, the two complementary strands of the parallel double helix have to be unwound
and are separated so that each can serve as a template for the synthesis of a new daughter strand.
Unwinding and the movement of replication for process occurs with the strands being transiently
unwound ahead of the fork for it moves along the chromosome. Three different types of proteins
appear to contribute to unwinding of the strands of double helices. (1) DNA unwinding of proteins or
DNA belicases are directly involved in catalyzing the unwinding of the double helices. E. coli, two
differ- ent helicases are involved. One helicase, the product of the rup gene, binds to and stimulates
separation the strand that has 3' to 5' polarity in the direction of replication fork movement. The
other helicase (exact identity still uncertain) binds to and assists unwinding of the strand that has 5'
to 3' polarity in the direction that the fork is moving (2) DNA single-strand bind ing proteins (SSBPs)
bind tightly to single-stranded regions of DNA produced by the action of the helicases and help
stabilize the extended single-stranded tem plates needed for polymerization. The SSBPs bind to NA
as tetramers, and their binding exhibits cooper tivity (ie, the binding of one tetramer stimulates the
binding of additional tetramers to adjacent segments of single-stranded DNA). The binding of SSBP
to single stranded DNA tends to hold that DNA in an extended configuration and prevents it from
folding back on itself. Single-stranded DNA that is saturated with bound SSBP replicates over 100
times faster than uncom plexed single-stranded DNA in vitro. Presumably, un complexed single
strands of DNA form secondary structures that interfere with the movement of DNA polymerases or
other components of the replication complex along the molecule in the normal processive manner.
(3) Finally, DNA gyrases, which catalyze the rmat'on of regative supercoils in DNA (see Fig 5.36), are
essential for replication and are believed to play a key role in the unwinding process. Supercoiling as
been proposed to help "drive" the unwinding process, however, we still do not know how this works
Very recently, it has been suggested that DNA gyrase may function by removing positive supercoils
that accumulate in front of the replication forks as the helicases unwind the double helices. In any
case, DNA gyrases are essential for DNA replication and someho maintain pre and postreplicative
DNAs in the proper topological structures.

Nascent DNA strands are initiated by the use of RNA primers by the mechanisın discussed earlier
(see Fig 5.29). The syntheses of the RNA primers are known as a special class of enzymes called
primases. Primase activity requires a complex of primase and at least six other proteins, this complex
is called the primosome. The primosome contains prepriming proteins, i n, n, n, and n "plus the
products of genes dnaB and dnaC (Table 5.4). The primosome carries out initial priming reaction for
the leading strand (the extended continuously strand in the over all 5 'to 3' direction) and the
repeating "Okazaki fragments" priming of synthesis for the lagging strand (synthesized
discontinuously in the overall 3 'to 5' butt direction 5 'to 3' at the molecular level, see Fig. 5.28) The
functions of individual proteins in the primosome are still uncertain The covalent extension (see Fig.
5.26) of the primed DNA chains during the chromosome replication in E. coli is carried out by DNA
polymerase III. Unlike DNA polymerase I of E. coli (which is a single poly peptide; see Fig. 5.25), DNA
polymerase II is complex enzyme containing seven different polypep tides (Fig. 5.31), and all of these
polyper tides present for proper replicative function. The 5 'to 3' polymerase activity and the 5 'to 3'
exonuclease activites are both present on the a polypeptide of DNA polymerase III. The 3 'to 5'
proofreading activity (see Fig. 5.27) of polymerase III is present on the e pol, peptide. The functions
of the other subunits are stil! uncertain. Subsequent to DNA polymerase II activity at the replication
fork, DNA polymerase I catalyzes the removal of the RNA primers by the 5 'to 3' concerted action
exonuclease activity and its 5 'to 3' polyasease activity, and DNA ligase catalyzes covalent sure of the
resulting single-stranded "nick" (Fig. 5.30). Several of the components of essential for DNA have
been identified genetically, that is, E coli strains carrying mutations (heritable changes in the genetic
material, see Chapter 11) that result in the inability to replicate DNA under certain conditions
(usually high temperature) have been identified. When these mutations were synthetized genetically
(see Chapters 7 and 8), they were found in a set of genes (designated dnaA, dnaB, etc.) whose
products were required for DNA synthesis in vivo. The products of these genes are known. For
example, dnaE, dnaN, dnax, and dnaZ the code for four of the seven subunits (polypeptides) of the
complete DNA polymerase III enzyme, and dnaG specifies the primase. The products and functions
of others (see Table 5.4) are still unknown. Other components of the replication enzymes have been
discovered by bioclave chemical analysis, and the exact functions of the many genes have not been
identified. products involved will be worked out during the next few years. Attempts to Isolate intact
functional replisomes, hovever, have been largely un successful. Reconstitution of subcoruplexes of
replica-application apparatuses from purified proteins has been more successful. This is undoubtedly
the result of the fact that complexes are held together by relatively weak isolation procedures. In
addition, replication of protein interactions, which are disrupted by durin comlexes may be
membrane-bound and require brane structures for their assembly. There is an evidence that is
associated with the cell membrane in projection and with the nuclear envelope in eukaryotes. For
excellent, more detailed accounts of the replication of the apparatus for the reader is DNA
replication and 1982 supplement to DNA replication. Phage (pX174 and "Rolling Circle" Replication
Bacteriophage pX174 are representative of small groups of viruses, both bacterial and eukaryotic,
that store their genetic information in single-stranded, circular molecule of DNA. When these viruses
infect a host cell, E coli in the case of px174, the single-stranded viral DNA (called the "positive" (+)
strand) is converted to a double helical form (called the "replicative form," RF) by the synthesis of a
complementary "negative" (-) strand. This double-stranded parental RF, which is replicated
asymmetrically to produce a large pop-ulation of viral (+) strands. The prognosis of viral strands are
then incorporated into protein coats to complete the reproductive cycle. The replication of the
Px174 chromosome can be divided into three stages: (1) parental (+) parental RF strand, (2) parental
RF projection RFs, and (3) projection RFs progeny (+) strands (Fig. 5.32) In the last two stages, DNA
synthesis mechanism occurs by the diffe rent mechanism the features of "rolling circulation" replica
d "shaped circle" rolling circle replication are the same as those discussed earlier for replication via
the more common 0, eye, image . This case, however, the replicative structure of a circular DNA
molecule with a single-stranded tail (Fig. 5.33) The sequence circle specific sequence of
endonuclease activity of the phage X174 gene A protein cleaves positive strand of the parental RF at
the origin of replication (Fig. 5.32). This endonuclease activity is site-specific; it cuts the PXI7
chromosome is only one site, the origin of replacements. It produces 3'-OH and 5'-phosphate
terminals at the cut in the (+) strand; the (-) strand remains intact. The 5 'end of the (+) strand is
wound and "peeled off while the (-) string rotates about its axis (thus the name" rolling circle "). This
yields the circle with its tail (Figs. 5.32 and 5.33), as originally proposed by W. Gilbert and D. Dressler,
called a "transferase, specific enzyme, called a" transferase, "which attached the 5 end of the (+)
strand to a specific site on cell membrane: Although most, if not all, replicating chromosomes are
attached to the mémbrane, little is known about the specific nature of attachments In any case, the
membrane attachment is an essential feature of rolling circle replication. rotates and the 5 'end is
displaced, DNA polymerase catalyzes covalent extension at the other (3'-OH) end.

During parental RF to RF RF replication, the wireless positive strands are used as templates for the
continuous synthesis of complementary negative strands. In some cases, the synthesis has been
completed. In such cases, a double-stranded tail will be produced. The switch from double-shifted RF
DNA synthesis to single-stranded viral (+) DNA sys- tems occurs when specific proteins of are
produced in the cell. Rolling cirdle replication continues, but as the viral strand is displaced, these
viral coats and strands (Fig. 532) the viral oX174 gene procedure is a key procedure in 4X174
replication. It possesses a remarkable activity (1) Gene A protein possesses a site-specific
endonuclease activity that keeps the positive strand at the origin. (2) Gene A protein then maintains
the energy of the cleaved phosphodiester linkage by means of a covalent attachment of the 5
'phosphoryl terminus to itself. (3) Gene A protein remains bound to the 5 min of the positive strand
for the traverse for the traverses the complete circular minus-strand template. (4) When a complete
positive strand has been synthesized, the gene A protein cleaves the new origin, ligates the 3'-
hydroxyl and 5'-phosphoryl termini, and once again becomes co-linked to the newly generated 5'-
positive terminal terminus. . This cycle of gene A protein activity until the population of RF (stage! 1)
or positive strands (stage Ill) is produced To date, evidence for rolling circle replication has been
found for (1) single-stranded DNA viruses like ox174, (2) the replication associated with some
transfer during conjugation in see (see Chapter 8), and (3) the replication of small genomic DNA in
the genes during oögenesis in amphibians ( see Chapter 15).