Rice University Lab Captures New Details of DNA Synthesis

Bioscientists in Texas have resolved a long-standing question about the role of metal ions in DNA synthesis, and they believe that the findings could help develop treatments for genetic mutations and the diseases they case, including cancer.

When cells reproduce, the internal mechanisms that copy DNA get it right nearly every time. But one of the ways replication can go wrong is known as misincorporation, which occurs when the order of nucleotides on the replicated DNA are in the incorrect order.  Using time-resolved crystallography, the team at Rice University (RU) found that a central metal ion critical to DNA replication also appears to be implicated in this faulty ordering of nucleotides.

RU structural biologist Yang Gao, graduate student Caleb Chang and alumna Christie Lee Luo analyzed polymerases, which are enzymes that bend and twist to rapidly reassemble complete strands of DNA. They specifically studied a polymerase known as eta, which is a translesion synthesis enzyme that guards against ultraviolet-induced lesions. People with mutations on the poly-eta gene often have a predisposition for xeroderma pigmentosum and skin cancer.

Until now, scientists could only guess at some details of the well-hidden mechanism by which polymerases do their job—and occasionally make mistakes. But the type of time-resolved crystallography used in Gao’s lab allowed the researchers to analyze proteins crystallized at 34 intermediate stages to define the positions of their atoms before, during and after DNA synthesis.

“This kinetic reaction is difficult to capture because there are many atoms, and they work very fast,” said Gao. “We’ve never known how the atoms move together because the spatial information was missing. Freezing the proteins and a small molecule substrate lets us capture this catalytic reaction for the first time.”

All proteins involved in DNA replication rely on metal ions—either magnesium or manganese—to catalyze the transfer of nucleotides to their proper positions along the strand, but whether there were two or three ions involved has long been a topic of debate. The RU team theorized that the first of the three metal atoms in eta supports nucleotide binding, and the second is the key to keeping the nucleotide and primer—short DNA strands that mark where polymerases start stringing new nucleotides—on track by stabilizing the binding of loose nucleotides to the primer located on the existing half of the new strand.  

“Only when the first two metal ions are in check can the third one come and drive the reaction home,” says Chang, who thinks the process may be universal among polymerases.

The researchers also noted poly-eta contains a motif that makes it prone to misalignment of primers, leading to a greater chance of misincorporation.

But for Gao, the real takeaway is in proving the ability of time-resolved crystallography to observe an entire catalytic process in atomic detail.  “This lets us see exactly what’s happening in a dynamic catalytic process over time,” he said.

 The findings were reported in Nature Communications.