KFU. Card publication. Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol

KAZAN
FEDERAL UNIVERSITY

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OPTIMIZATION OF THE EXPRESSION, PURIFICATION AND POLYMERASE ACTIVITY REACTION CONDITIONS OF RECOMBINANT HUMAN PRIMPOL

Form of presentation

Articles in international journals and collections

Year of publication

2017

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Authors, employees of KFU

Khayrullin Rafil Fidailevich, author

Other authors

Wanrooij Sjoerd , author

Bibliographic description in the original language

Boldinova EO, Stojkovič G, Khairullin R, Wanrooij S, Makarova AV (2017) Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol. PLoS ONE 12(9): e0184489. https://doi. org/10.1371/journal.pone.0184489

Annotation

Human PrimPol is a DNA primase/polymerase involved in DNA damage tolerance and prevents nuclear genome instability. PrimPol is also localized to the mitochondria, but its precise function in mitochondrial DNA maintenance has remained elusive. PrimPol works both as a translesion (TLS) polymerase and as the primase that restarts DNA replication after a lesion. However, the observed biochemical activities of PrimPol vary considerably between studies as a result of different reaction conditions used. To reveal the effects of reaction composition on PrimPol DNA polymerase activity, we tested the polymerase activity in the presence of various buffer agents, salt concentrations, pH values and metal cofactors. Additionally, the enzyme stability was analyzed under various conditions. We demonstrate that the reaction buffer with pH 6–6.5, low salt concentrations and 3 mM Mg²⁺ or 0.3–3 mM Mn²⁺ cofactor ions supports the highest DNA polymerase activity of human PrimPol in vitro. The DNA polymerase

Keywords

PrimPol, DNA polymerase, mitochondria, protein expression.

The name of the journal

PLos ONE

URL

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0184489

Please use this ID to quote from or refer to the card

https://repository.kpfu.ru/eng/?p_id=163498&p_lang=2

Resource files

File name	Size (MB)	Format
Optimization_of_the_expression_purification_and_po.pdf	6,71	pdf	show / download

Full metadata record

Field DC	Value	Language
dc.contributor.author	Khayrullin Rafil Fidailevich	ru_RU
dc.contributor.author	Wanrooij Sjoerd	ru_RU
dc.date.accessioned	2017-01-01T00:00:00Z	ru_RU
dc.date.available	2017-01-01T00:00:00Z	ru_RU
dc.date.issued	2017	ru_RU
dc.identifier.citation	Boldinova EO, Stojkovič G, Khairullin R, Wanrooij S, Makarova AV (2017) Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol. PLoS ONE 12(9): e0184489. https://doi. org/10.1371/journal.pone.0184489	ru_RU
dc.identifier.uri	https://repository.kpfu.ru/eng/?p_id=163498&p_lang=2	ru_RU
dc.description.abstract	PLos ONE	ru_RU
dc.description.abstract	Human PrimPol is a DNA primase/polymerase involved in DNA damage tolerance and prevents nuclear genome instability. PrimPol is also localized to the mitochondria, but its precise function in mitochondrial DNA maintenance has remained elusive. PrimPol works both as a translesion (TLS) polymerase and as the primase that restarts DNA replication after a lesion. However, the observed biochemical activities of PrimPol vary considerably between studies as a result of different reaction conditions used. To reveal the effects of reaction composition on PrimPol DNA polymerase activity, we tested the polymerase activity in the presence of various buffer agents, salt concentrations, pH values and metal cofactors. Additionally, the enzyme stability was analyzed under various conditions. We demonstrate that the reaction buffer with pH 6–6.5, low salt concentrations and 3 mM Mg²⁺ or 0.3–3 mM Mn²⁺ cofactor ions supports the highest DNA polymerase activity of human PrimPol in vitro. The DNA polymerase	ru_RU
dc.language.iso	ru	ru_RU
dc.subject	PrimPol	ru_RU
dc.subject	DNA polymerase	ru_RU
dc.subject	mitochondria	ru_RU
dc.subject	protein expression.	ru_RU
dc.title	Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol	ru_RU
dc.type	Articles in international journals and collections	ru_RU