In this article we will discuss about a generalised model of DNA replication in prokaryotes.
DNA replication is catalyzed by the enzyme DNA polymerase, and it involves several other proteins/enzyme.
The data collected from both in vitro and in vivo studies have been synthesized into a generalized model DNA replication, a brief and simplified description of which is given below (Fig. 28.5):
1. DNA replication begin at certain unique and fixed points called ‘origin’.
2. Two enzymes DNA gyrase and DNA helicase, bind to the origin points and induce the unwinding complementary strands of DNA double helix; this is known as DNA melting (Fig. 28.6).
3. Melting produces two Y-shaped forks at origin; one fork is located at each end of the origin. When replication begins, these forks become replication forks (Fig. 28.6).
4. Certain proteins, called single-strand binding proteins, bind to the single-stranded regions the produced. As a result, these regions remain single-stranded.
5. An enzyme called primase initiates transcription of the strand whose 3′-end is single-stranded (3’→ strand); this generates a 10-60 nucleotide long primer RNA (transcribed in 5’→ 3′ direction).
6. The free 3′-OH of this primer RNA provides the initiation point for DNA ploymerase for the sequential addition of deoxyribonucleotides. DNA polymerase has an absolute requirement of a free 3’ -OH of a pre-existing polynucleotide for the initiation of DNA replication. DNA polymera progressively adds deoxyribonucleotides to the free 3′-OH of this growing polynucleotide chai. Consequently, the replication of 3’→ 5′ strand of a DNA molecule proceeds continuously.
7. The replication of second strand (5’→ 3′ strand) of the DNA molecule is dis-continuous, and begin somewhat later than that of the 3’→ 5′ strand. Therefore the 3′ → 5′ strand of a DNA molecule known as the leading strand, while the 5′ → 3′ strand is termed as the lagging strand.
8. When replication of the 3′ → 5′ strand has progressed for some time, primase initiates the synthesis RNA primer on the 5’→ 3′ strand close to the replication fork (away from the origin). The prim synthesis begins close to the replication fork and progresses towards the origin.
The 3′-OH of the primer RNA provides the initiation point for DNA polymerase to catalyze replication of the ‘lagging strand’. Obviously, the replication of lagging strand proceeds from the replication fork towards the origin, i.e., its direction is opposite to that of the leading strand.
9. After some more time of replication of the leading (3′ → 5′) strand, primase again initiates primer synthesis on the 5′ → 3′ (lagging) strand near the fork. The primer RNA thus formed provides the free 3′-OH for replication of the single-stranded region of the lagging strand.
10. Clearly, replication of the lagging (5′ → 3′) strand generates small polynucleotide fragments called ‘Okazaki fragments’. The replication of this strand is discontinuous in that it has to be initiated several times, and every times one Okazaki fragment is produced.
11. The RNA primer associated with the newly synthesized DNA strands/Okazaki fragments is most likely digested by DNA polymerase I in prokaryotes. This enzyme also catalyzes the filling of gaps so generated in the new strands. The Okazaki fragments (after the gap-filling by DNA polymerase I) are joined together by the enzyme polynucleotide ligase.
12. The above description pertains to the DNA replication progressing in one direction from the origin; the same events also occur in the opposite direction of the origin.
Variations from the Generalized Scheme:
Variations in DNA replication occur in the manner of replication initiation, the scheme of replication fork movement and the timings of the replication of the two strands of the DNA molecule.
These variations are represented in the following models of DNA replication:
(1) Strand displacement,
(2) Rolling circle and
(3) D-loop mechanism of DNA replication.
(1) Strand Displacement DNA Replication:
In this scheme, replication begins at one end of a linear genome (= chromosome), e.g., in adenoviruses. Only one of the two strands, the strand oriented in 3′ → 5′ direction, of the DNA duplex is replicated in a continuous manner. This produces a DNA duplex and a single strand (the 5′ → 3′ strand).
The single strand base pairs at its ends to from a circle: replication of this strand now proceeds in a continuous manner (Fig. 28.7). In adenovirus, a protein attached to the 5′-end provides the primer function. This protein has a cytosine residue attached to it; the free 3′-OH of this C is used as primer.
(2) Rolling Circle Replication:
Rolling circle replication occurs in many small bacterial and eukaryotic viruses, e.g., in bactriophage φX174 φ, Greek letter ‘phi’ here stands for phage). φX174 has a single stranded genome, i.e., the plus-strand, which is converted through discontinuous replication into the double-stranded replicative form (RF).
Phage protein A induces a nick, i.e., break in a single strand, in one strand (the + strand) of the double-stranded RF chromosome. The nick occurs at the origin and protein A binds to the 5′-end so produced. The 3′-end produced at the nick serves as primer and DNA synthesis proceeds from this end.
As a result, the + strand is progressively displaced from the (-) strand beginning at the 5′-end. Replication produces a single strand copy of the + strand, which functions as the viral genome. When a complete positive strand has been synthesized, and a new origin is produced in the (+) strand, protein A nicks in the origin to produce one single strand copy of the phage genome (Fig. 28.8).
(3) Displacement Loop DNA Replication:
This mode of replication occurs in mammalian mitochondria; the mitochondrial chromosome in circular. One strand of the chromosome is denoted as H strand, while its complementary strand is named L strand. Replication begins at a specific origin, but only one strand, the H strand, is replicated; the other strand (the L strand) of the chromosome is displaced forming a loop, called displacement loop or D loop.
When replication of (the H strand) has progressed up to about 2/3 of the chromosome, the replication of the displaced single strand (L strand) begins.
This replication begins at a different origin and replication proceeds in the opposite direction. Thus both the strands of DNA are replicated in a continuous manner, their replications begin at different origins, and replication of one strand (H strand) begins much earlier than that of the other (L strand).
In this case, replication is not only unidirectional, but the replication of only one of the two strands takes place at each of the replication forks (Fig. 28.9). The same is the case with rolling circle replication.