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Title: Phosphate steering by Flap Endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability

Abstract

DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5'-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5'-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 5'polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via phosphate steering', basic residues energetically steer an inverted ss 5'-flap through a gateway over FEN1's active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA) n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5'-flap specificity and catalysis, preventing genomic instability.

Authors:
 [1];  [2];  [3];  [4];  [2];  [2];  [4];  [2];  [2];  [2]; ORCiD logo [5];  [1];  [6]; ORCiD logo [6];  [4];  [2]; ORCiD logo [7]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  2. Univ. of Sheffield (United Kingdom). Centre for Chemical Biology, Sheffield Inst. for Nucleic Acids (SInFoNiA), Dept. of Chemistry
  3. Scripps Research Inst., La Jolla, CA (United States). Dept. of Molecular Biology
  4. Tufts Univ., Medford, MA (United States). Dept. of Biology
  5. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering
  6. King Abdullah Univ. of Science and Technology, Thuwal (Saudi Arabia). Division of Biological and Environmental Sciences and Engineering
  7. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of Texas, Houston, TX (United States). Dept. of Molecular and Cellular Oncology
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
National Institutes of Health (NIH); USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1408438
Grant/Contract Number:
AC02-05CH11231; AC02-76SF00515
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 8; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES

Citation Formats

Tsutakawa, Susan E., Thompson, Mark J., Arvai, Andrew S., Neil, Alexander J., Shaw, Steven J., Algasaier, Sana I., Kim, Jane C., Finger, L. David, Jardine, Emma, Gotham, Victoria J. B., Sarker, Altaf H., Her, Mai Z., Rashid, Fahad, Hamdan, Samir M., Mirkin, Sergei M., Grasby, Jane A., and Tainer, John A. Phosphate steering by Flap Endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability. United States: N. p., 2017. Web. doi:10.1038/ncomms15855.
Tsutakawa, Susan E., Thompson, Mark J., Arvai, Andrew S., Neil, Alexander J., Shaw, Steven J., Algasaier, Sana I., Kim, Jane C., Finger, L. David, Jardine, Emma, Gotham, Victoria J. B., Sarker, Altaf H., Her, Mai Z., Rashid, Fahad, Hamdan, Samir M., Mirkin, Sergei M., Grasby, Jane A., & Tainer, John A. Phosphate steering by Flap Endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability. United States. doi:10.1038/ncomms15855.
Tsutakawa, Susan E., Thompson, Mark J., Arvai, Andrew S., Neil, Alexander J., Shaw, Steven J., Algasaier, Sana I., Kim, Jane C., Finger, L. David, Jardine, Emma, Gotham, Victoria J. B., Sarker, Altaf H., Her, Mai Z., Rashid, Fahad, Hamdan, Samir M., Mirkin, Sergei M., Grasby, Jane A., and Tainer, John A. Tue . "Phosphate steering by Flap Endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability". United States. doi:10.1038/ncomms15855. https://www.osti.gov/servlets/purl/1408438.
@article{osti_1408438,
title = {Phosphate steering by Flap Endonuclease 1 promotes 5'-flap specificity and incision to prevent genome instability},
author = {Tsutakawa, Susan E. and Thompson, Mark J. and Arvai, Andrew S. and Neil, Alexander J. and Shaw, Steven J. and Algasaier, Sana I. and Kim, Jane C. and Finger, L. David and Jardine, Emma and Gotham, Victoria J. B. and Sarker, Altaf H. and Her, Mai Z. and Rashid, Fahad and Hamdan, Samir M. and Mirkin, Sergei M. and Grasby, Jane A. and Tainer, John A.},
abstractNote = {DNA replication and repair enzyme Flap Endonuclease 1 (FEN1) is vital for genome integrity, and FEN1 mutations arise in multiple cancers. FEN1 precisely cleaves single-stranded (ss) 5'-flaps one nucleotide into duplex (ds) DNA. Yet, how FEN1 selects for but does not incise the ss 5'-flap was enigmatic. Here we combine crystallographic, biochemical and genetic analyses to show that two dsDNA binding sites set the 5'polarity and to reveal unexpected control of the DNA phosphodiester backbone by electrostatic interactions. Via phosphate steering', basic residues energetically steer an inverted ss 5'-flap through a gateway over FEN1's active site and shift dsDNA for catalysis. Mutations of these residues cause an 18,000-fold reduction in catalytic rate in vitro and large-scale trinucleotide (GAA)n repeat expansions in vivo, implying failed phosphate-steering promotes an unanticipated lagging-strand template-switch mechanism during replication. Thus, phosphate steering is an unappreciated FEN1 function that enforces 5'-flap specificity and catalysis, preventing genomic instability.},
doi = {10.1038/ncomms15855},
journal = {Nature Communications},
number = ,
volume = 8,
place = {United States},
year = {Tue Jun 27 00:00:00 EDT 2017},
month = {Tue Jun 27 00:00:00 EDT 2017}
}

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  • No abstract prepared.
  • Human flap endonuclease 1 complexed with nicked DNA has been crystallized. A diffraction data set was collected to a resolution of 2.75 Å. Flap endonuclease 1 (FEN1) is a structure-specific nuclease that removes the RNA/DNA primer associated with Okazaki fragments in DNA replication. Here, crystals of the complex between the catalytic domain of human FEN1 and a DNA product have been obtained. For efficient crystallization screening, a DNA–protein complex crystallization screening (DPCS) kit was designed based on commercial crystallization kits. The crystal was found to belong to space group P2{sub 1}, with unit-cell parameters a = 61.0, b = 101.3,more » c = 106.4 Å, β = 106.4°. The asymmetric unit is predicted to contain two complexes in the crystallographic asymmetric unit. A diffraction data set was collected to a resolution of 2.75 Å.« less
  • Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never missesmore » cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.« less
    Cited by 1
  • Human flap endonuclease 1 (FEN1) and related structure-specific 5’nucleases precisely identify and incise aberrant DNA structures during replication, repair and recombination to avoid genomic instability. Yet, it is unclear how the 5’nuclease mechanisms of DNA distortion and protein ordering robustly mediate efficient and accurate substrate recognition and catalytic selectivity. Here, single-molecule sub-millisecond and millisecond analyses of FEN1 reveal a protein-DNA induced-fit mechanism that efficiently verifies substrate and suppresses off-target cleavage. FEN1 sculpts DNA with diffusion-limited kinetics to test DNA substrate. This DNA distortion mutually ‘locks’ protein and DNA conformation and enables substrate verification with extreme precision. Strikingly, FEN1 never missesmore » cleavage of its cognate substrate while blocking probable formation of catalytically competent interactions with noncognate substrates and fostering their pre-incision dissociation. These findings establish FEN1 has practically perfect precision and that separate control of induced-fit substrate recognition sets up the catalytic selectivity of the nuclease active site for genome stability.« less
    Cited by 1