Published October 19, 2015
Research led by Mark Sutton, PhD, professor of biochemistry, has described a new model for DNA polymerase switching in E. coli that will better the understanding of antibiotic resistance and genetic mutations.
All organisms suffer from DNA damage. Cells can either repair this damage, tolerate the damage or do nothing and die.
DNA damage tolerance leads to mutations that underlie genetic diseases in humans. These mutations also lead to antibiotic resistance and adaption of bacterial pathogens in their human hosts.
Sutton and Michelle K. Scotland, a candidate in the Department of Biochemistry’s doctoral program, studied how cells coordinate the actions of their translesion synthesis (TLS) DNA polymerases with their replicative DNA polymerases.
“The fundamentals of studying the mechanisms of this bacteria will later be applicable to the mechanisms of human cells. Although some important differences exist, the fundamental need to coordinate the actions of these different polymerases is conserved throughout evolution,” explained Sutton.
In addition to accurately copying damaged DNA, TLS polymerases are also able to replicate undamaged DNA, but they do so with low accuracy.
Consequently, the actions of TLS polymerases must be tightly controlled to limit mutations while still enabling cells to copy damaged DNA.
Using E. coli as a model, the researchers focused on studying coordinate regulation of Pol III, the replicative DNA polymerase, and Pol IV, a TLS polymerase, with a focus on the beta sliding clamp, a protein that tethers DNA polymerases to the DNA template.
“Every time E. coli encounters a damaged site, it has to either repair itself or develop a genetic mutation. Humans and other organisms have to do the same thing,” said Sutton.
Pol IV is conserved throughout evolution, making it an ideal model to understand how TLS polymerases are regulated.
Mutations catalyzed by Pol IV are critically important for antibiotic resistance and adaptation of bacterial pathogens.
“Pol IV is required for bacteria to undergo mutations and antibiotic resistance. If we can figure out a way to block its access to Pol III, we could apply that knowledge to other adapted mutations and antibiotic resistance,” said Sutton.
Understanding how Pol IV gains access to the replication fork to catalyze mutations will help develop new therapies to control its function.
In collaboration with Harvard University’s Loparo Lab and Cleveland State University’s Berdis Lab, Scotland determined that the Pol IV-T120P mutation interferes with the ability of Pol IV to displace Pol III from the beta clamp in vitro and in vivo.
The research shows that both a Poll III-Pol IV interaction and a Pol IV-beta clamp interaction are required for Poll III-Pol IV switching.
Scotland determined that Pol IV switches with Pol III at DNA damaged sites by first binding the Pol III-beta clamp complex. Then Pol IV displaces Pol III from the clamp to ultimately gain control of the replication fork to catalyze potentially mutagenic TLS.
This represents a new model for Pol III-Pol IV switching that has strong biochemical and genetic support and likely applies to other polymerase switches.
Understanding how these switch mechanisms work is of fundamental importance, said Sutton.
“Every organism that’s been examined has one or more polymerases important for DNA replication and one or more to tolerate damage. The actions of both polymerases need to be perfectly coordinated with each other in all cells,” he said.