Step 3: Synthesis of leading and lagging strands

The double strands of template DNA are antiparallel and ‘run’ in opposite directions, but the DNA copying machine (DNA pol III) can only work in one direction. This means there will be continuous (leading) and discontinuous (lagging) replication happening at each replication fork (Fig. 13c). 

Figure 13c. Each replication fork has its own leading and lagging strand due to the antiparallel nature of DNA. At each helicase, one strand opened for reading has 3’ to 5’ orientation and the other has 5’ to 3’ orientation. DNA pol III can continuously read in the 3’ to 5’ direction, but when information is opened in the 5’ to 3’ direction, more primers are needed to help DNA pol III copy the DNA strand. Credit: M. Sutter, 2024.

Leading strand formation: The strand that can be replicated on a continuous basis as helicase unwinds the DNA is called the leading strand. In figure 13c, there are two leading strands, one for each fork. For the left fork, the leading strand of DNA is being created from the bottom strand since the helicase is opening up 3’ to 5’ DNA for DNA pol III to read in the 3’ to 5’ direction. The other leading strand is found at the top strand going to the right. This is because after grabbing onto a primer, DNA pol III can continually read in the 3’ to 5’ direction as helicase opens the DNA that is in the 3’ to 5’ orientation. 

Lagging strand formation: The strand that cannot be replicated on a continuous basis is called the lagging strand. Let’s focus on just the right replication fork in figure 13c that is orange colored to learn about this principle. Notice how the anti-parallel nature of DNA means that the helicase on the right opens up 3’ to 5’ information on the top DNA strand (the leading strand) while simultaneously opening up 5’ to 3’ information on the bottom DNA strand. This bottom strand needs to be copied, but it is impossible for DNA pol III to read in the 5’ to 3’ direction. It is possible for DNA pol III to make a copy of this strand using it’s 3’ to 5’ reading direction, but many more primers are required to form multiple for DNA pol III to read the strand. The product of this process of adding more RNA primers to allow DNA pol III to copy the strand of DNA opening in the 5’ to 3’ direction creates “fragments” of DNA (green lines) intermixed with RNA primers (pink lines). 

This same phenomenon of a lagging strand happens for the left replication fork on the top strand. This is because the left replication fork on the top strand is opening up a single strand that is in the 5’ to 3’ direction and DNA pol III can only read in the 3’ to 5’ direction, again resulting in the need to place multiple primers to complete the copying process by DNA pol III. Because replication always moves away from the advancing replication fork, this new strand is the “lagging strand”. 


Figure 13d. Short fragments made by discontinuous replication were discovered by the Okazaki team and are called the Okazaki fragments. Credit: W. Suza, 2021.

One of the first scientists to provide evidence to support this model of DNA replication was the team of Reiji and Tsuneko Okazaki in the 1960s. This is why these short lagging strand segments of the lagging strand are called “Okazaki fragments” (Fig. 13d). They used radioactive nucleotides to track the newly replicating fragments and found that many of the fragments were short. This fits with the discontinuous model. 

Figure 13e. DNA pol I replaces RNA primers with DNA nucleotides. In this image, two primers (top right & one on the bottom right) have been replaced. DNA pol I still needs to replace the RNA primers on the top left and bottom right with DNA. Credit: M. Sutter, 2024.  

For both the leading and lagging strands, DNA pol I removes the RNA nucleotide primers and replaces them with DNA nucleotides (Fig. 13e).  

As the four enzymes just described attend to their specific roles in replication, two double stranded DNA molecules are created, each with an old (template) strand and a newly formed strand. If the DNA pol III enzyme makes a mistake and adds the wrong, non-complementing nucleotide, the enzyme can proofread its work and replace these replication mistakes. When replication is perfect, the two double stranded DNA molecules will have identical sequences.