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

Susan E. Tsutakawa; Mark J. Thompson; Andrew S. Arvai; Alexander J. Neil; Steven J. Shaw; Sana I. Algasaier; Jane C. Kim; L. David Finger; Emma Jardine; Victoria J.B. Gotham; Altaf H. Sarker; Mai Z. Her; Fahad Rashid; Samir Hamdan; Sergei M. Mirkin; Jane A. Grasby; John A. Tainer

doi:10.1038/ncomms15855

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

Susan E. Tsutakawa, Mark J. Thompson, Andrew S. Arvai, Alexander J. Neil, Steven J. Shaw, Sana I. Algasaier, Jane C. Kim, L. David Finger, Emma Jardine, Victoria J.B. Gotham, Altaf H. Sarker, Mai Z. Her, Fahad Rashid, Samir Hamdan, Sergei M. Mirkin, Jane A. Grasby, John A. Tainer

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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) 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.

Original language	English (US)
Journal	Nature Communications
Volume	8
Issue number	1
DOIs	https://doi.org/10.1038/ncomms15855
State	Published - Jun 27 2017

Bibliographical note

KAUST Repository Item: Exported on 2020-10-01
Acknowledgements: For financial support, we thank NCI (PO1CA92584 to J.A.T.); BBSRC (grants BB/K009079/1 and BB/M00404X/1 to J.A.G.); the Ministry of Higher Education Libya, EPSRC and the University of Sheffield (studentships to S.I.A., S.J.S. and E.J., respectively); NIH (R01GM060987 to S.M. and R01GM110387 to S.T.); and KAUST for core and CRG3 funding to S.M.H. and J.A.T. J.A.T. acknowledges support by a Robert A. Welch Chemistry Chair, the Cancer Prevention and Research Institute of Texas, and the University of Texas System Science and Technology Acquisition and Retention. The tumour mutation analysis includes data generated by the TCGA Research Network: http://cancergenome.nih.gov/. We thank the ALS, SSRL, the IDAT program, the DOE BER and the NIH project MINOS (R01GM105404) for X-ray data facilities. We thank James Holton for aiding X-ray data analysis.

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10.1038/ncomms15855

https://repository.kaust.edu.sa/bitstream/10754/625186/1/ncomms15855.pdf

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Tsutakawa, S. E., Thompson, M. J., Arvai, A. S., Neil, A. J., Shaw, S. J., Algasaier, S. I., Kim, J. C., Finger, L. D., Jardine, E., Gotham, V. J. B., Sarker, A. H., Her, M. Z., Rashid, F., Hamdan, S., Mirkin, S. M., Grasby, J. A., & Tainer, J. A. (2017). Phosphate steering by Flap Endonuclease 1 promotes 5′-flap specificity and incision to prevent genome instability. Nature Communications, 8(1). https://doi.org/10.1038/ncomms15855

@article{74f92aab09eb4d4e8abfb5a3d2209b7a,

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) 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.",

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 Emma Jardine and Gotham, {Victoria J.B.} and Sarker, {Altaf H.} and Her, {Mai Z.} and Fahad Rashid and Samir Hamdan and Mirkin, {Sergei M.} and Grasby, {Jane A.} and Tainer, {John A.}",

note = "KAUST Repository Item: Exported on 2020-10-01 Acknowledgements: For financial support, we thank NCI (PO1CA92584 to J.A.T.); BBSRC (grants BB/K009079/1 and BB/M00404X/1 to J.A.G.); the Ministry of Higher Education Libya, EPSRC and the University of Sheffield (studentships to S.I.A., S.J.S. and E.J., respectively); NIH (R01GM060987 to S.M. and R01GM110387 to S.T.); and KAUST for core and CRG3 funding to S.M.H. and J.A.T. J.A.T. acknowledges support by a Robert A. Welch Chemistry Chair, the Cancer Prevention and Research Institute of Texas, and the University of Texas System Science and Technology Acquisition and Retention. The tumour mutation analysis includes data generated by the TCGA Research Network: http://cancergenome.nih.gov/. We thank the ALS, SSRL, the IDAT program, the DOE BER and the NIH project MINOS (R01GM105404) for X-ray data facilities. We thank James Holton for aiding X-ray data analysis.",

year = "2017",

month = jun,

day = "27",

doi = "10.1038/ncomms15855",

language = "English (US)",

volume = "8",

journal = "Nature Communications",

issn = "2041-1723",

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Tsutakawa, SE, Thompson, MJ, Arvai, AS, Neil, AJ, Shaw, SJ, Algasaier, SI, Kim, JC, Finger, LD, Jardine, E, Gotham, VJB, Sarker, AH, Her, MZ, Rashid, F, Hamdan, S, Mirkin, SM, Grasby, JA & Tainer, JA 2017, 'Phosphate steering by Flap Endonuclease 1 promotes 5′-flap specificity and incision to prevent genome instability', Nature Communications, vol. 8, no. 1. https://doi.org/10.1038/ncomms15855

TY - JOUR

T1 - Phosphate steering by Flap Endonuclease 1 promotes 5′-flap specificity and incision to prevent genome instability

AU - Tsutakawa, Susan E.

AU - Thompson, Mark J.

AU - Arvai, Andrew S.

AU - Neil, Alexander J.

AU - Shaw, Steven J.

AU - Algasaier, Sana I.

AU - Kim, Jane C.

AU - Finger, L. David

AU - Jardine, Emma

AU - Gotham, Victoria J.B.

AU - Sarker, Altaf H.

AU - Her, Mai Z.

AU - Rashid, Fahad

AU - Hamdan, Samir

AU - Mirkin, Sergei M.

AU - Grasby, Jane A.

AU - Tainer, John A.

N1 - KAUST Repository Item: Exported on 2020-10-01 Acknowledgements: For financial support, we thank NCI (PO1CA92584 to J.A.T.); BBSRC (grants BB/K009079/1 and BB/M00404X/1 to J.A.G.); the Ministry of Higher Education Libya, EPSRC and the University of Sheffield (studentships to S.I.A., S.J.S. and E.J., respectively); NIH (R01GM060987 to S.M. and R01GM110387 to S.T.); and KAUST for core and CRG3 funding to S.M.H. and J.A.T. J.A.T. acknowledges support by a Robert A. Welch Chemistry Chair, the Cancer Prevention and Research Institute of Texas, and the University of Texas System Science and Technology Acquisition and Retention. The tumour mutation analysis includes data generated by the TCGA Research Network: http://cancergenome.nih.gov/. We thank the ALS, SSRL, the IDAT program, the DOE BER and the NIH project MINOS (R01GM105404) for X-ray data facilities. We thank James Holton for aiding X-ray data analysis.

PY - 2017/6/27

Y1 - 2017/6/27

N2 - 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) 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.

AB - 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) 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.

UR - http://hdl.handle.net/10754/625186

UR - https://www.nature.com/articles/ncomms15855

UR - http://www.scopus.com/inward/record.url?scp=85021400624&partnerID=8YFLogxK

U2 - 10.1038/ncomms15855

DO - 10.1038/ncomms15855

M3 - Article

C2 - 28653660

SN - 2041-1723

VL - 8

JO - Nature Communications

JF - Nature Communications

IS - 1

ER -

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

Abstract

Bibliographical note

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