All posts by Brian LM Cheng

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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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).


A visit to the current controversy: manmade mutant H5N1 influenza virus (1)

A researcher, named Ron Fouchier, claimed that he has created a new variant (or you may call it the mutant) of H5N1 influenza virus. Normally, H5N1 influenza virus can not be transmitted between human and human. And it rarely transmits from an avian source to human. But when it does, the fatality rate is approximately 50%. Dr. Fouchier, who is an investigator at the Erasmus Medical Center in the Netherlands, claimed that he has created a new and airborne H5N1 influenza virus. This mutant is transmissible between mammals. Although his research finding is yet published and opened to the public, his research has created a controversy in the balance of academic freedom and protecting the nation from bioterrorism. As a member of the general public, I, too, have no idea what the introduced mutation Dr. Fouchier made to the original H5N1 strain. As a nascent virologist here at UC, here I will make some speculation and discuss about the ongoing situation.

Redefining the risk of mutant virus

One of the key aspects circulating in the news is the fact that this mutant is “airborne”. In my opinion, almost all viruses that disseminate within the respiratory tract are airborne. If viruses are able to replicate in our lung, it is highly possible that the mucus or droplets from an infected individual contain a considerable amount of viruses. The reason why some viruses is not able to disseminate effectively is possibly due to the failure to replicate efficiently within the host, resulting in a low concentration/titer of viruses which may not be enough to infect another susceptible individual.

“TONY EASTLEY: Scientists who have produced an airborne mutated of the killer bird flu virus H5N1 say it’s essential their research be published, but opponents say the biosecurity risks are too high and would be a “how to” manual for terrorists.” ABC NEWS

The public is now informed that this mutant virus is airborne. But honestly, the concern should be focus on the fact that it has now adapted to be spread between human and human. In the past, humans are the accidental host of H5N1 viruses. In other words, it is rare when the H5N1 viruses infect humans. So the concern should focus on the fact that the specific mutation can change the targeting species and disseminating efficiency. For an effective virus replication cycle, the virus must find a susceptible host cell that expresses a certain protein on its membrane. These proteins are known as receptors. These receptors are the key for the virus to recognize the proper cells to enter. The next barrier the virus needs to overcome is whether the host cells can provide the necessary factors for it to replicate.

In the next issue, we will continue to discuss how the viral surface proteins can be mutated to recognize different hosts (which I think it’s one of the key barriers to derive a new species in viruses). We will go over some current literature on CHIKV mutation and how the mutation on the viral surface protein can change the selectivity and the replication efficiency.


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A recurring theme: virus exploits the host’s machinery.

One of the most fascinating thing I found associated with all types of viruses is that they are capable of hijacking cellular machinery with only a few genes. In the case of dengue virus (DENV), it only codes for 7 nonstructural and 3 structural genes. And how these tiny viruses can take over host cells of about 20000-25000 genes is just fascinating. I attended a seminar, called “The ESCRT pathway in HIV budding and cell division” today down at GCIS. It was hosted by Dr. Wesley Sundquist from University of Utah (shown right). And here, I will talk about what the major concepts mentioned in the talk.

ImageAccording to wikipedia, “ESCRT (endosomal sorting complex required for transport) refers to a series of cytosolic protein complexes called ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III.” There are 3 major roles of ESCRT machinery: (1) multivesicular bodies (MVB) formation, (2) virus budding, and (3) cytokinesis. The MVB formation is important in recycling receptors that are endocytosed into the cells. One of the prime examples is the EGF receptor that gets endocytosed after binding to the EGF molecules. After the EGFR is internalized in the lysosome, it is then localized in the MVB to traffick to the endosomes for ubiquitination, which is then expressed on the cell surface for another round of binding to EGFR. In the case of cytokinesis, ESCRT-III forms a multimer around the midbody between two daughter cells (in other words, it wrap around the junction between two daughter cells). Then, an ATPase interacts with ESCRT-III to facilitate the cleavage and separate the two daughter cells. (it is likely that this ATPase contribute a conformational change to the ESCRT-III proteins, which give rise to enough thermal energy to break apart the junction.

HIV exploits this pinching technique by ESCRT-III to bud off from the host cells. It was shown that the envelope protein, Gag, interacts with ALIX. ALIX then interacts with ESCRT-III, which then Vps4 ATPase comes in to facilitate the pinching. As a result, progeny HIV can then be released from the host cells to continue infect neighboring uninfected cells. In the case of ALIX-depleted (siRNA-ALIX treated) cells (see below), HIV is not able to bud well from the host cell, and there is some distinguishable difference in the envelope of the HIV between the wildtype HIV and ALIX binding site mutant HIV.

Taken together, HIV exploits the cellular cleavage mechanism used for cytokinesis to facilitate its budding from the host.



– Virus exploits cellular pathways to facilitate its replication cycle

– Cellular ESCRT pathway is important for HIV budding.


Fujii et al. Freed, Functional role of Alix in HIV-1 replication, Virology, Volume 391, Issue 2, 1 September 2009, 284-292, J. Virol.

Wollert et al. The ESCRT machinery at a glance. July 1, 2009 J Cell Sci 122, 2163-2166.

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