MSGID: 1:317/3 6279eac4   
   PID: hpt/lnx 1.9.0-cur 2019-01-08   
   TID: hpt/lnx 1.9.0-cur 2019-01-08   
    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.   
      
      
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   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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