Crystal study may resolve DNA mystery
Lab captures unseen details of replication, clues to how mutations can
happen

Date:
May 9, 2022
Source:
Rice University
Summary:
Bioscientists have uncovered a tiny detail that could help us
understand how DNA replicates with such astounding accuracy.



FULL STORY ==========================================================================
When cells reproduce, the internal mechanisms that copy DNA get it right
nearly every time. Rice University bioscientists have uncovered a tiny
detail that helps us understand how the process could go wrong.


========================================================================== Their study of enzymes revealed the presence of a central metal
ion critical to DNA replication also appears to be implicated in misincorporation, the faulty ordering of nucleotides on new strands.

The observation reported in Nature Communications could help find
treatments for genetic mutations and the diseases they cause, including
cancer.

Rice structural biologist Yang Gao, graduate student Caleb Chang and
alumna Christie Lee Luo used time-resolved crystallography to analyze
the flexible enzymes called polymerase as they bend and twist to
rapidly reassemble complete strands of DNA from a pool of C, G, A and
T nucleotides.

All of the 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 Rice team seems to have settled that through studying a polymerase
known as eta, a translesion synthesis enzyme that guards against ultraviolet-induced lesions. Those with mutations on the poly-eta gene
often have a predisposition for xeroderma pigmentosum and skin cancer, according to the researchers.



==========================================================================
Gao said typical polymerases resemble a right-handed shape, and he thinks
of them in terms of an actual hand: "They have a palm domain that holds
the active site, a finger domain that closes up to interact with the
new base pair, and a thumb domain that binds the primer/template DNA,"
he said.

But until now, scientists could only guess at some details of the
well-hidden mechanism by which polymerases do their job, and occasionally
fail. 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, an assistant professor of biosciences who joined Rice as a CPRIT Scholar in 2019. "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." The study led to
their theory 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 on track by stabilizing the binding of loose nucleotides
to the primer located on the existing half of the new strand (aka the substrate). Primers are short DNA strands that mark where polymerases
start stringing new nucleotides.

"Only when the first two metal ions are in check can the third one come
and drive the reaction home," said Chang, suggesting 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.

"This is, first, about a basic mechanism of life," Gao said. "DNA has to
be copied accurately, and errors 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 there's a very limited amount of
energy available for them to choose the right base pair." 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 Cancer Prevention and Research Institute of Texas (RR190046), the
Welch Foundation (C-2033-20200401) and a predoctoral fellowship from
the Houston Area Molecular Biophysics Program (National Institutes of
Health grant T32 GM008280) supported the research.


========================================================================== Story Source: Materials provided by Rice_University. Original written
by Mike Williams. Note: Content may be edited for style and length.


========================================================================== Journal Reference:
1. Caleb Chang, Christie Lee Luo, Yang Gao. In crystallo observation of
three metal ion promoted DNA polymerase misincorporation. Nature
Communications, 2022; 13 (1) DOI: 10.1038/s41467-022-30005-3 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2022/05/220509171059.htm

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