Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase e{open}

Walter J. Zahurancik, Andrey G. Baranovskiy, Tahir H Tahirov, Zucai Suo

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Numerous genetic studies have provided compelling evidence to establish DNA polymerase e{open} (Pole{open}) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Pole{open} is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3'. →. 5' exonuclease domain common to many replicative polymerases. In addition, Pole{open} possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Pole{open} heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Pole{open} in vitro. However, similar studies of the human Pole{open} heterotetramer (hPole{open}) have been limited by the difficulty of obtaining hPole{open} in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPole{open} from insect host cells has allowed for isolation of greater amounts of active hPole{open}, thus enabling a more detailed kinetic comparison between hPole{open} and an active N-terminal fragment of the hPole{open} catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPole{open}. We observe that the small subunits increase DNA binding by hPole{open} relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3'. →. 5' exonuclease activity of hPole{open} is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPole{open} and sway hPole{open} toward DNA synthesis rather than proofreading.

Original languageEnglish (US)
Pages (from-to)16-22
Number of pages7
JournalDNA Repair
Volume29
DOIs
StatePublished - May 1 2015

Fingerprint

Holoenzymes
DNA-Directed DNA Polymerase
Kinetic parameters
Poles
Catalytic Domain
DNA
Phosphodiesterase I
Human Activities
Catalyst activity
Saccharomycetales
Baculoviridae
DNA Replication
Insects
Yeast
Escherichia coli
Purification
Maintenance
Genome
Genes

Keywords

  • 3'→5' exonuclease activity
  • DNA polymerase epsilon
  • Human DNA replication
  • Leading strand replication
  • Pre-steady-state kinetics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase e{open}. / Zahurancik, Walter J.; Baranovskiy, Andrey G.; Tahirov, Tahir H; Suo, Zucai.

In: DNA Repair, Vol. 29, 01.05.2015, p. 16-22.

Research output: Contribution to journalArticle

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abstract = "Numerous genetic studies have provided compelling evidence to establish DNA polymerase e{open} (Pole{open}) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Pole{open} is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3'. →. 5' exonuclease domain common to many replicative polymerases. In addition, Pole{open} possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Pole{open} heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Pole{open} in vitro. However, similar studies of the human Pole{open} heterotetramer (hPole{open}) have been limited by the difficulty of obtaining hPole{open} in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPole{open} from insect host cells has allowed for isolation of greater amounts of active hPole{open}, thus enabling a more detailed kinetic comparison between hPole{open} and an active N-terminal fragment of the hPole{open} catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPole{open}. We observe that the small subunits increase DNA binding by hPole{open} relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3'. →. 5' exonuclease activity of hPole{open} is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPole{open} and sway hPole{open} toward DNA synthesis rather than proofreading.",
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N2 - Numerous genetic studies have provided compelling evidence to establish DNA polymerase e{open} (Pole{open}) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Pole{open} is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3'. →. 5' exonuclease domain common to many replicative polymerases. In addition, Pole{open} possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Pole{open} heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Pole{open} in vitro. However, similar studies of the human Pole{open} heterotetramer (hPole{open}) have been limited by the difficulty of obtaining hPole{open} in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPole{open} from insect host cells has allowed for isolation of greater amounts of active hPole{open}, thus enabling a more detailed kinetic comparison between hPole{open} and an active N-terminal fragment of the hPole{open} catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPole{open}. We observe that the small subunits increase DNA binding by hPole{open} relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3'. →. 5' exonuclease activity of hPole{open} is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPole{open} and sway hPole{open} toward DNA synthesis rather than proofreading.

AB - Numerous genetic studies have provided compelling evidence to establish DNA polymerase e{open} (Pole{open}) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Pole{open} is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3'. →. 5' exonuclease domain common to many replicative polymerases. In addition, Pole{open} possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Pole{open} heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Pole{open} in vitro. However, similar studies of the human Pole{open} heterotetramer (hPole{open}) have been limited by the difficulty of obtaining hPole{open} in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPole{open} from insect host cells has allowed for isolation of greater amounts of active hPole{open}, thus enabling a more detailed kinetic comparison between hPole{open} and an active N-terminal fragment of the hPole{open} catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPole{open}. We observe that the small subunits increase DNA binding by hPole{open} relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3'. →. 5' exonuclease activity of hPole{open} is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPole{open} and sway hPole{open} toward DNA synthesis rather than proofreading.

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