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Spotlight articles: Trading Places on DNA (Yuzhakov et al. 1999) Review

Today I will briefly discuss one of the landmark papers in molecular biology. This paper is by Alexander Yuzhakov, Zvi Kelman and Mike O’Donnell, from the Rockefeller University and the HHMI in New York City.

DNA duplex has an elegant structure, yet its replication relies on a sophisticated and complex network of factors. During DNA replication, helicase unwinds the DNA duplex. SSB binds to this single stranded region to maintain and stabilize the structure. On the lagging strand, primase is required to produce a series of RNA primers. The β sliding clamp is then loaded onto the primed site to enable the extension by DNA polymerase III. This landmark paper, by O’Donnell group, tries to understand how primase is displaced from DNA. They hypothesize that there is a component in the DNA replication that removes primase after RNA primer is made. This paper is highly significant as (i) it shows that there is a competition between components to enable efficient and regulated DNA replication; (ii) and due to this competition, components that are present at a low concentration, such as primase, can be recycled.

This paper started from trying to understand the temperature-sensitive replication defect on phage DNA when it is coated with SSB-113 mutant. When phage G4 ssDNA is primed with synthetic RNA, both SSB- and SSB-113-coated ssDNA can replicate at permissive and restrictive temperatures. However, at restrictive temperature, SSB-113-coated ssDNA can not be replicated. This defect is not due to the malfunction of primase. Knowing that the β-clamp is loaded after primase priming, they then continue to see if the loading is defective. Using 32P-labeled β-clamp, they realize that the clamp loading is defective in SSB-113-coated ssDNA at restrictive temperature, which suggests at restrictive temperature, the SSB-113 prohibits the clamp loading, which may consequently leads to replication defect. They then continue to ask what causes the displacement of primase. β-clamp alone does not displace primase, but DNA Pol III and the β-γ complex does. Interestingly, χ subunit of Pol III alone displaces primase effectively. Because Pol III does not displace primase at restrictive temperature when ssDNA is coated with SSB-113, they then hypothesize χ’s interaction with SSB is temperature sensitive. χ does not interact with primase. When ssDNA is coated with SSB-113, χ shows decreased interaction comparing to SSB-coated ssDNA. In contrast, primase does not show this interaction discrepancy with SSB-113. Interestingly, as χ increases, primase bound to the ssDNA decreases, which indicates that there is a competition between χ subunit and primase for SSB interaction. To confirm the competition, they compare the level of synthesized DNA and bound primase in the presence and absence of χ. In the absence of χ, primase remains bound to the ssDNA, and replication of lagging strand is defective. In the presence of χ, primase is displaced, and lagging strand is replicated. Last but not the least, they show that the displacement by Pol III is polymerase-specific, as other polymerases do not replicate. This primase-polymerase switch is also species-specific as human clamp PCNA does not load when phage primase is present. The primase also protects the RNA primer from RNAse. Taken together, primase-polymerase switch depends on the competition between χ subunit of RNAP and primase for interacting with SSB. Primase also protects the RNA primer, and its displacement from SSB-coated ssDNA is species- and polymerase-specific.

Mike O’Donnell’s group has presented an important mechanism in DNA replication with well-supported evidence. The paper’s successful attempt to bring the switch concept in phage G4 ssDNA into a broader and general concept, such as replication fork, is admirable. This brilliant attempt is shown in Fig. 5, with well thought-out designs to prove the displacement function of χ subunit. O’Donnell’s approach to answer the questions is logical, and I believe that this paper has answered the questions posed. However, the author has yet addressed why the primase is not displaced at restrictive temperature. On 154, the author states, “the χ subunit of Pol III holoenzyme interacts much more weakly with SSB-113 than with wild-type SSB”. It is possible that the res. 113 is essential for interacting with χ. And at restrictive temperature, interaction with res. 113 is lost possibly due to conformational change. SSB-113 carries Pro (non-polar) to Ser (polar) mutation. To see if the polarity of this residue is important for interaction with χ, we can mutate the Pro113 to Cys113 (slightly polar). By immobilizing the Cys113 mutant in SPR, we can see if this slightly-polar residue would result in intermediate interaction. Fig. 1A illustrates how the primase produces RNA primer in a 5′-3′ direction. How does χ detect the primase-bound SSB? As χ does not interact with primase (Fig. 4C), does primase binding change the structure of SSB providing a signal for χ subunit recognition? To address this question, we have to first understand where χ binds to in ssDNA. In other words, does χ bind to the opposite or the same face of primase? To approach, we can perform DMS methylation footprinting to probe for MG/mg regions that are protected by χ and primase. By comparing the binding regions, we will have a better understanding on how the displacement works.

 

 

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Reference:

Yuzhakov A., Kelman Z., O’Donnell M. (1999). Trading places on DNA — a three point switch underlies primer handoff from primase to the replicative DNA polymerase. Cell (96, 153-163).