New Insight into Metal Ion-Driven Catalysis of Nucleic Acids by Influenza PA-Nter.
PA subunit of influenza RNA-dependent RNA polymerase deserves constantly increasing attention due to its essential role in influenza life cycle. N-terminal domain of PA (PA-Nter) harbors endonuclease activity, which is indispensable in viral transcription and replication. Interestingly, existing lit...
Main Authors: | , |
---|---|
Format: | Article |
Language: | English |
Published: |
Public Library of Science (PLoS)
2016-01-01
|
Series: | PLoS ONE |
Online Access: | http://europepmc.org/articles/PMC4907508?pdf=render |
id |
doaj-25c821373fc54bbeba3378fdfd8131c6 |
---|---|
record_format |
Article |
spelling |
doaj-25c821373fc54bbeba3378fdfd8131c62020-11-25T01:48:04ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-01116e015697210.1371/journal.pone.0156972New Insight into Metal Ion-Driven Catalysis of Nucleic Acids by Influenza PA-Nter.Daria KotlarekRemigiusz WorchPA subunit of influenza RNA-dependent RNA polymerase deserves constantly increasing attention due to its essential role in influenza life cycle. N-terminal domain of PA (PA-Nter) harbors endonuclease activity, which is indispensable in viral transcription and replication. Interestingly, existing literature reports on in vitro ion preferences of the enzyme are contradictory. Some show PA-Nter activity exclusively with Mn2+, whereas others report Mg2+ as a natural cofactor. To clarify it, we performed a series of experiments with varied ion concentrations and substrate type. We observed cleavage in the presence of both ions, with a slight preference for manganese, however PA-Nter activity highly depended on the amount of residual, co-purified ions. Furthermore, to quantify cleavage reaction rate, we applied fluorescence cross-correlation spectroscopy (FCCS), providing highly sensitive and real-time monitoring of single molecules. Using nanomolar ssDNA in the regime of enzyme excess, we estimated the maximum reaction rate at 0.81± 0.38 and 1.38± 0.34 nM/min for Mg2+ and Mn2+, respectively. However, our calculations of PA-Nter ion occupancy, based on thermodynamic data, suggest Mg2+ to be a canonical metal in PA-Nter processing of RNA in vivo. Presented studies constitute a step toward better understanding of PA-Nter ion-dependent activity, which will possibly contribute to new successful inhibitor design in the future.http://europepmc.org/articles/PMC4907508?pdf=render |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Daria Kotlarek Remigiusz Worch |
spellingShingle |
Daria Kotlarek Remigiusz Worch New Insight into Metal Ion-Driven Catalysis of Nucleic Acids by Influenza PA-Nter. PLoS ONE |
author_facet |
Daria Kotlarek Remigiusz Worch |
author_sort |
Daria Kotlarek |
title |
New Insight into Metal Ion-Driven Catalysis of Nucleic Acids by Influenza PA-Nter. |
title_short |
New Insight into Metal Ion-Driven Catalysis of Nucleic Acids by Influenza PA-Nter. |
title_full |
New Insight into Metal Ion-Driven Catalysis of Nucleic Acids by Influenza PA-Nter. |
title_fullStr |
New Insight into Metal Ion-Driven Catalysis of Nucleic Acids by Influenza PA-Nter. |
title_full_unstemmed |
New Insight into Metal Ion-Driven Catalysis of Nucleic Acids by Influenza PA-Nter. |
title_sort |
new insight into metal ion-driven catalysis of nucleic acids by influenza pa-nter. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2016-01-01 |
description |
PA subunit of influenza RNA-dependent RNA polymerase deserves constantly increasing attention due to its essential role in influenza life cycle. N-terminal domain of PA (PA-Nter) harbors endonuclease activity, which is indispensable in viral transcription and replication. Interestingly, existing literature reports on in vitro ion preferences of the enzyme are contradictory. Some show PA-Nter activity exclusively with Mn2+, whereas others report Mg2+ as a natural cofactor. To clarify it, we performed a series of experiments with varied ion concentrations and substrate type. We observed cleavage in the presence of both ions, with a slight preference for manganese, however PA-Nter activity highly depended on the amount of residual, co-purified ions. Furthermore, to quantify cleavage reaction rate, we applied fluorescence cross-correlation spectroscopy (FCCS), providing highly sensitive and real-time monitoring of single molecules. Using nanomolar ssDNA in the regime of enzyme excess, we estimated the maximum reaction rate at 0.81± 0.38 and 1.38± 0.34 nM/min for Mg2+ and Mn2+, respectively. However, our calculations of PA-Nter ion occupancy, based on thermodynamic data, suggest Mg2+ to be a canonical metal in PA-Nter processing of RNA in vivo. Presented studies constitute a step toward better understanding of PA-Nter ion-dependent activity, which will possibly contribute to new successful inhibitor design in the future. |
url |
http://europepmc.org/articles/PMC4907508?pdf=render |
work_keys_str_mv |
AT dariakotlarek newinsightintometaliondrivencatalysisofnucleicacidsbyinfluenzapanter AT remigiuszworch newinsightintometaliondrivencatalysisofnucleicacidsbyinfluenzapanter |
_version_ |
1725013191781515264 |