Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate.

Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate. Research Abstract Details 

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Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate. Abstract Text:

Flap endonuclease 1 (FEN1) is a structure-specific nuclease that cleaves substrates containing unannealed 5'-flaps during Okazaki fragment processing. Cleavage removes the flap at or near the point of annealing. The preferred substrate for archaeal FEN1 or the 5'-nuclease domains of bacterial DNA polymerases is a double-flap structure containing a 3'-tail on the upstream primer adjacent to the 5'-flap. We report that FEN1 in Saccharomyces cerevisiae (Rad27p) exhibits a similar specificity. Cleavage was most efficient when the upstream primer contained a 1-nucleotide 3'-tail as compared with the fully annealed upstream primer traditionally tested. The site of cleavage was exclusively at a position one nucleotide into the annealed region, allowing human DNA ligase I to seal all resulting nicks. In contrast, a portion of the products from traditional flap substrates is not ligated. The 3'-OH of the upstream primer is not critical for double-flap recognition, because Rad27p is tolerant of modifications. However, the positioning of the 3'-nucleotide defines the site of cleavage. We have tested substrates having complementary tails that equilibrate to many structures by branch migration. FEN1 only cleaved those containing a 1-nucleotide 3'-tail. Equilibrating substrates containing 12-ribonucleotides at the end of the 5'-flap simulates the situation in vivo. Rad27p cleaves this substrate in the expected 1-nucleotide 3'-tail configuration. Overall, these results suggest that the double-flap substrate is formed and cleaved during eukaryotic DNA replication in vivo.

Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate. Publishing Authors By Initials

For similar biochemical phenomena, metabolism, and nutrition: biochemical phenomena: substrate specificity research abstracts see: biochemical phenomena, metabolism, and nutrition: biochemical phenomena: substrate specificity research

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Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate. Journal Published:

PUBLICATION TYPE: Research Support, U.S. Gov't,

Journal: The Journal of biological chemistry

VOLUME: 277

Page Numbers: 14379-89

Journal Abbreviation: J. Biol. Chem.

ISSN: 0021-9258

DAY: 1

MONTH: 02

YEAR: 2002

Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate. Information

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LANGUAGE: eng

NlmUniqueID: 2985121

Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate. Keywords Mesh Terms:

KEYWORDS: Substrate Specificity

MESH TERMS: enzymology

Chemical & Substance for Abstract: Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate. Information

Substance Name: FEN1 protein, human

Registry Number: EC 3.1.11.5

Grant and Affiliation Information for Cleavage specificity of Saccharomyces cerevisiae flap endonuclease 1 suggests a double-flap structure as the cellular substrate.

AFFILIATION: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.

Country: United States

AGENCY: United States NIGMS

GRANT: GM24441

ACRONYM: GM

MEDLINETA: J Biol Chem

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