HOUSTON, TX – When cells reproduce, the internal mechanisms that copy DNA succeed almost every time. Rice University bioscientists have discovered a small detail that helps understand how the process could go wrong.
Their study of the enzymes revealed the presence of a central metal ion essential to DNA replication also appears to be involved in the misincorporation, the faulty order of nucleotides on new strands.
The sighting reported in Nature Communication could help find treatments for genetic mutations and the diseases they cause, including cancer.
Structural rice biologist Yang Gao, graduate student Caleb Chang, and alumnus Christie Lee Luo used time-resolved crystallography to analyze flexible enzymes called polymerases as they bend and twist to rapidly reassemble complete strands of DNA from a pool of C, G, A and T nucleotides.
All proteins involved in DNA replication rely on metal ions – magnesium or manganese – to catalyze the transfer of nucleotides to their proper positions along the strand, but the question of whether there were two or three ions involved has long been a matter of debate.
Rice’s team appears to have settled this by studying a polymerase known as eta, a translesion-synthesizing enzyme that protects against ultraviolet-induced damage. According to the researchers, people with mutations in the poly-eta gene often have a predisposition to xeroderma pigmentosum and skin cancer.
Gao said typical polymerases resemble a right-handed shape, and he views them in terms of the actual hand: “They have a palm domain that contains the active site, a finger domain that closes to interact with the new pair of bases, and a thumb domain that binds primer/template DNA,” he said.
But until now, scientists could only guess at certain details of the well-hidden mechanism by which polymerases do their job, and sometimes fail. The type of time-resolved crystallography used in Gao’s lab allowed researchers to analyze crystallized proteins at 34 intermediate steps to define the positions of their atoms before, during and after DNA synthesis.
“This kinetic reaction is hard to capture because there are a lot of atoms and they are moving very fast,” said Gao, an assistant professor of biosciences who joined Rice as a CPRIT fellow in 2019. “We never knew how atoms move together because spatial information was missing Freezing proteins and a small molecule substrate allows us to capture this catalytic reaction for the first time.
The study led to their theory that the first of eta’s three metal atoms supports nucleotide binding, and the second is the key to keeping the nucleotide and primer on track by stabilizing free nucleotide binding. to the primer located on the existing half of the new strand (aka the substrate). Primers are short strands of DNA that mark where polymerases begin to string together new nucleotides.
“Only when the first two metal ions are in check can the third one come in and drive the reaction home,” Chang said, suggesting the process could be universal among polymerases.
The researchers also noted that poly-eta contains a motif that makes it prone to primer misalignment, which increases the chances of misincorporation.
“It is, first, a basic mechanism of life,” Gao said. “DNA must be copied precisely, and mistakes can lead to human disease. People who study these enzymes know that for DNA synthesis, they always do much, much better than they should, because the amount of energy available to choose the right base pair is very limited.
For Gao, the real takeaway is to prove the ability of time-resolved crystallography to observe an entire catalytic process in atomic detail.
“It allows us to see exactly what is happening in a dynamic catalytic process over time,” he said.
– This press release was originally published on the Rice University website