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

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:
Grant/Contract Number:
AC02-05CH11231; AC02-76SF00515
Type:
Accepted Manuscript
Journal Name:
Nature Communications
Additional Journal Information:
Journal Volume: 8; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
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)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES
OSTI Identifier:
1408438

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., 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.. 2017. "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 = {2017},
month = {6}
}

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