Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A.N. Ogurtsov 2011

DNA Replication and Repair
Bidirectional Replication

As shown in Figures 53 and 54, after unwinding and Separation by the Replication fork, both parental strands are copied into daughter strands.

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Figure 55 - Bidirectional METABOLISM/36.html">DNA replication

A single origin can initiate either a single replication event—where the replication fork moves in one direction along the parental helix—or bidirectional replication (Figure 55), in which two replication forks are established at the same origin and move in opposite directions. Bidirectional growth has been observed in numerous experiments, including the one presented in Figure 55.

The circular viral DNA was cleaved with the restriction enzyme EcoRI, and the replication region was then observed to expand symmetrically in both directions from the origin, demonstrating bidirectional replication.

It is now widely accepted that all prokaryotic and Eukaryotic Cells utilize the bidirectional replication process.

Thus, in the previously discussed example, bidirectional replication of SV40 DNA is initiated by the binding of two large T-antigen helicases to a single SV40 origin, followed by the recruitment of other essential Proteins to form two replication forks.

These two forks then move in opposite directions away from the origin. As shown in Figure 56, the left replication fork synthesizes DNA from right to left, and the right fork from left to right.

Figure 56 - Scheme of bidirectional DNA replication

Utilizing the energy of ATP Hydrolysis, the helicases move in opposite directions, generating two single-stranded chains stabilized by RPA proteins (Figure 56, step 1).

Primase-Pol a complexes synthesize short primers on each of the parental template strands (Figure 56, step 2).

PCNA-Rfc-Pol d complexes replace the primase-Pol a complexes and extend the short primers, forming the leading strand at each replication fork (Figure 56, step 3).

The helicases continue to unwind the duplex, and RPA proteins bind to the newly unwound single-stranded regions (Figure 56, step 4).

PCNA-Rfc-Pol d complexes continue to extend the leading strands (Figure 56, step 5).

Primase-Pol a complexes synthesize primers for lagging strand synthesis at both replication forks (Figure 56, step 6).

PCNA-Rfc-Pol d complexes replace the primase-Pol a complexes and extend the Okazaki fragments of the lagging strands, which are eventually joined to the 5' ends of the leading strands. The ligation site is marked by a circle (Figure 56, step 7).

Replication proceeds through the further repetition of steps 4–7, i.e., DNA unwinding and the synthesis of leading and lagging strands.

Since the origin region is copied into the daughter strands first during replication, the initiation of a new replication round is possible even before the completion of the previous one, giving rise to the branched Structure shown in Figure 57.

This "dichotomous" replication allows Bacteria under favorable conditions to have a genome generation time shorter than the time required to complete a round of DNA replication.

Unlike SV40 DNA, eukaryotic chromosomal DNA molecules contain multiple replication origins separated by hundreds and thousands of kilobases (kb). A six-subunit protein called the origin recognition complex (ORC) binds to each origin and recruits the proteins required to load cellular hexameric helicases—consisting of six homologous MCM proteins—onto the DNA.

Figure 57 - Dichotomous replication of a bacterial chromosome

Two oppositely directed MCM helicases unwind the parental duplex at the origin. RPA (replication protein A) proteins bind to the separated single strands, optimally orienting them for daughter strand synthesis. Primer synthesis and subsequent steps in cellular DNA replication are currently believed to proceed in the same manner as in SV40 DNA replication (Figures 53 and 54).

Cellular DNA Replication and other events leading to Cell proliferation are finely regulated to ensure that the requisite number of cells for each tissue is produced during the Organism's growth and lifespan. Just as in the Transcription of most genes, initiation control serves as the primary regulatory mechanism for cellular DNA replication. The activation of MCM helicases, which is essential for initiating DNA replication, is regulated by specific protein Kinases known as S-phase cyclin-dependent kinases. Other cyclin-dependent kinases regulate additional aspects of Cell Division, including the complex process of mitosis by which eukaryotic cells divide into two daughter cells.