Question

Sliding clamps are loaded onto DNA by clamp loaders to serve the critical role of coordinating various enzymes on DNA. Clamp loaders must quickly and efficiently load clamps at primer/template (p/t) junctions containing a duplex region with a free 3′OH (3′DNA), but it is unclear how clamp loaders target these sites. To measure the Escherichia coli and Saccharomyces cerevisiae clamp loader specificity toward 3′DNA, fluorescent β and PCNA clamps were used to measure clamp closing triggered by DNA substrates of differing polarity, testing the role of both the 5′phosphate (5′P) and the presence of single-stranded binding proteins (SSBs). SSBs inhibit clamp loading by both clamp loaders on the incorrect polarity of DNA (5′DNA). The 5′P groups contribute selectivity to differing degrees for the two clamp loaders, suggesting variations in the mechanism by which clamp loaders target 3′DNA. Interestingly, the χ subunit of the E. coli clamp loader is not required for SSB to inhibit clamp loading on phosphorylated 5′DNA, showing that χ·SSB interactions are dispensable. These studies highlight a common role for SSBs in directing clamp loaders to 3′DNA, as well as uncover nuances in the mechanisms by which SSBs perform this vital role.”

https://academic.oup.com/nar/article/42/16/10655/2903350

(5 points) What is a sliding clamp and what is its function?

(5 points) What is a sliding clamp loader and how does it aid in the function of the sliding clamp?

(5 points) What is the significance of a recessed 3’-end DNA, during the p/t discrimination? What data and figure from the NAR paper, suggest this significance?

(5 points) What do you think would happen if the sliding clamp could not differentiate between 3’-OH and 5’-P ends of DNA? How would this effect DNA replication?

Answer 1

Sliding clamps and clamp loaders were found to have a greater role in DNA replication and repair processes. Sliding clamps are circular proteins that are observed to be helping the DNA polymerase to get attached and replicate the template DNA. Without the sliding clamps, DNA polymerase would get dissociated from DNA frequently hindering the replication process.

Clamp loaders are enzymes that catalyze the sliding clamp attachment to the DNA template during the replication process. The rate of DNA synthesis is increased with the presence of sliding clamps on the template DNA.

The clamp loaders need ATP to bind to the sliding clamps with the DNA. There are two types of clamp loaders discussed in this article. The E. Coli loader is called gamma complex heptamer and S. cerevisiae clamp loader is replication factor C which is a pentamer. These are ATPase enzymes that are cup shaped.

DNA binding stimulates ATP hydrolysis leading to the detachment of DNA-clamp complex. For stabilizing interactions with DNA, proteins like DNA polymerase and others need sliding clamps and hence these clamps are needed to be placed exactly in a particular location. The location of clamps have to be precise as their faces should allow protein-protein interactions to take place without any problem. For instance, DNA polymerases will find and bind the sliding clamps at the primer –template junction having recessed 3’ ends to carryout DNA replication and repair. If the sliding clamp is not attached in that particular orientation at that location, the polymerase cannot bind to the DNA and carryout the replication process effectively to synthesize DNA. It is observed that a fresh sliding clamp is required for every 2 to 3 seconds on the lagging strand for every Okazaki fragment. The clamp loader has to recognize the primer-template junction efficiently and quickly to load a sliding clamp.

In the investigation on the specificity of clamp loaders for 3’ end of DNA to carry out the replication effectively, it is observed that 100-fold difference was there between 3’ DNA and 5’ DNA, conferring DNA p/t junction specificity to the gamma complex of the clamp loader. This is due to the presence of SSB at the 3’ end. The polarity information of the DNA that is compatible to the clamp loader to carry out the clamping is conferred effectively by SSBs to the clamp loader gamma component. The test was done with 400nm SSB which is known to reduce the clamp closing rate on 5’ DNA.SSB by 100-fold compared to that at the 3’DNA.SSB end.