Monitoring the Retention of Human Proliferating Cell Nuclear Antigen at Primer/Template Junctions by Proteins That Bind Single-Stranded DNA

Mark Hedglin, Mahesh Aitha, Stephen Benkovic

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

In humans, proliferating cell nuclear antigen (PCNA) sliding clamps encircling DNA coordinate various aspects of DNA metabolism throughout the cell cycle. A critical aspect of this is restricting PCNA to the vicinity of its DNA target site. For example, PCNA must be maintained at or near primer/template (P/T) junctions during DNA synthesis. With a diverse array of cellular factors implicated, many of which interact with PCNA, DNA, or both, it is unknown how this critical feat is achieved. Furthermore, current biochemical assays that examine the retention of PCNA near P/T junctions are inefficient, discontinuous, and qualitative and significantly deviate from physiologically relevant conditions. To overcome these challenges and limitations, we recently developed a novel and convenient Förster resonance energy transfer (FRET) assay that directly and continuously monitors the retention of human PCNA at a P/T junction. Here we describe in detail the design, methodology, interpretation, and limitations of this quantitative FRET assay using the single-stranded DNA-binding protein, SSB, from Escherichia coli as an example. This powerful tool is broadly applicable to any single-stranded DNA-binding protein and may be utilized and/or expanded upon to dissect DNA metabolic pathways that are dependent upon PCNA.

Original languageEnglish (US)
Pages (from-to)3415-3421
Number of pages7
JournalBiochemistry
Volume56
Issue number27
DOIs
StatePublished - Jul 11 2017

Fingerprint

Single-Stranded DNA
Proliferating Cell Nuclear Antigen
Monitoring
DNA
Proteins
Assays
Energy Transfer
DNA-Binding Proteins
Energy transfer
Clamping devices
Metabolic Networks and Pathways
Metabolism
Escherichia coli
Cell Cycle
Cells

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{99cedee621ac46c3af749c4fbdf8a917,
title = "Monitoring the Retention of Human Proliferating Cell Nuclear Antigen at Primer/Template Junctions by Proteins That Bind Single-Stranded DNA",
abstract = "In humans, proliferating cell nuclear antigen (PCNA) sliding clamps encircling DNA coordinate various aspects of DNA metabolism throughout the cell cycle. A critical aspect of this is restricting PCNA to the vicinity of its DNA target site. For example, PCNA must be maintained at or near primer/template (P/T) junctions during DNA synthesis. With a diverse array of cellular factors implicated, many of which interact with PCNA, DNA, or both, it is unknown how this critical feat is achieved. Furthermore, current biochemical assays that examine the retention of PCNA near P/T junctions are inefficient, discontinuous, and qualitative and significantly deviate from physiologically relevant conditions. To overcome these challenges and limitations, we recently developed a novel and convenient F{\"o}rster resonance energy transfer (FRET) assay that directly and continuously monitors the retention of human PCNA at a P/T junction. Here we describe in detail the design, methodology, interpretation, and limitations of this quantitative FRET assay using the single-stranded DNA-binding protein, SSB, from Escherichia coli as an example. This powerful tool is broadly applicable to any single-stranded DNA-binding protein and may be utilized and/or expanded upon to dissect DNA metabolic pathways that are dependent upon PCNA.",
author = "Mark Hedglin and Mahesh Aitha and Stephen Benkovic",
year = "2017",
month = "7",
day = "11",
doi = "10.1021/acs.biochem.7b00386",
language = "English (US)",
volume = "56",
pages = "3415--3421",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "27",

}

Monitoring the Retention of Human Proliferating Cell Nuclear Antigen at Primer/Template Junctions by Proteins That Bind Single-Stranded DNA. / Hedglin, Mark; Aitha, Mahesh; Benkovic, Stephen.

In: Biochemistry, Vol. 56, No. 27, 11.07.2017, p. 3415-3421.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Monitoring the Retention of Human Proliferating Cell Nuclear Antigen at Primer/Template Junctions by Proteins That Bind Single-Stranded DNA

AU - Hedglin, Mark

AU - Aitha, Mahesh

AU - Benkovic, Stephen

PY - 2017/7/11

Y1 - 2017/7/11

N2 - In humans, proliferating cell nuclear antigen (PCNA) sliding clamps encircling DNA coordinate various aspects of DNA metabolism throughout the cell cycle. A critical aspect of this is restricting PCNA to the vicinity of its DNA target site. For example, PCNA must be maintained at or near primer/template (P/T) junctions during DNA synthesis. With a diverse array of cellular factors implicated, many of which interact with PCNA, DNA, or both, it is unknown how this critical feat is achieved. Furthermore, current biochemical assays that examine the retention of PCNA near P/T junctions are inefficient, discontinuous, and qualitative and significantly deviate from physiologically relevant conditions. To overcome these challenges and limitations, we recently developed a novel and convenient Förster resonance energy transfer (FRET) assay that directly and continuously monitors the retention of human PCNA at a P/T junction. Here we describe in detail the design, methodology, interpretation, and limitations of this quantitative FRET assay using the single-stranded DNA-binding protein, SSB, from Escherichia coli as an example. This powerful tool is broadly applicable to any single-stranded DNA-binding protein and may be utilized and/or expanded upon to dissect DNA metabolic pathways that are dependent upon PCNA.

AB - In humans, proliferating cell nuclear antigen (PCNA) sliding clamps encircling DNA coordinate various aspects of DNA metabolism throughout the cell cycle. A critical aspect of this is restricting PCNA to the vicinity of its DNA target site. For example, PCNA must be maintained at or near primer/template (P/T) junctions during DNA synthesis. With a diverse array of cellular factors implicated, many of which interact with PCNA, DNA, or both, it is unknown how this critical feat is achieved. Furthermore, current biochemical assays that examine the retention of PCNA near P/T junctions are inefficient, discontinuous, and qualitative and significantly deviate from physiologically relevant conditions. To overcome these challenges and limitations, we recently developed a novel and convenient Förster resonance energy transfer (FRET) assay that directly and continuously monitors the retention of human PCNA at a P/T junction. Here we describe in detail the design, methodology, interpretation, and limitations of this quantitative FRET assay using the single-stranded DNA-binding protein, SSB, from Escherichia coli as an example. This powerful tool is broadly applicable to any single-stranded DNA-binding protein and may be utilized and/or expanded upon to dissect DNA metabolic pathways that are dependent upon PCNA.

UR - http://www.scopus.com/inward/record.url?scp=85023203454&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85023203454&partnerID=8YFLogxK

U2 - 10.1021/acs.biochem.7b00386

DO - 10.1021/acs.biochem.7b00386

M3 - Article

C2 - 28590137

AN - SCOPUS:85023203454

VL - 56

SP - 3415

EP - 3421

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 27

ER -