Interaction and Stimulation of Human FEN-1 Nuclease Activities by Heterogeneous Nuclear Ribonucleoprotein A1 in α-Segment Processing during Okazaki Fragment Maturation

Qing Chai, Li Zheng, Mian Zhou, John Turchi, Binghui Shen

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

High-fidelity DNA replication depends on both accurate incorporation of nucleotides in the newly synthesized strand and the maturation of Okazaki fragments. In eukaryotic cells, the latter is accomplished by a series of coordinated actions of a set of structure-specific nucleases, which, with the assistance of accessory proteins, recognize branched RNA/DNA configurations. In the current model of Okazaki fragment maturation, displacement of a 27-nucleotide or longer flap is envisioned to attract replication protein A (RPA), which inhibits flap endonuclease-1 (FEN-1) but stimulates Dna2 nuclease for cleavage. Dna2 cleavage generates a short flap of 5-7 nucleotides, which resists binding by RPA and further cleavage by Dna2. FEN-1 then removes the remaining flap to produce a suitable substrate for ligation. However, FEN-1 is not efficient in cleaving the short flap, and we therefore set out to identify cellular factors that might regulate FEN-1 activity. Through co-immunoprecipitation experiments, we have isolated heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), which forms a direct complex with FEN-1 and stimulates its enzymatic activities. The stimulation by hnRNP A1 is most dramatic using DNA substrates with short flaps. With longer flap substrates the hnRNP A1 effect is more modest and is suppressed by the addition of RPA. A model is provided to explain the possible in vivo role of this interaction and activity in Okazaki fragment maturation.

Original languageEnglish (US)
Pages (from-to)15045-15052
Number of pages8
JournalBiochemistry
Volume42
Issue number51
DOIs
StatePublished - Dec 30 2003
Externally publishedYes

Fingerprint

Flap Endonucleases
Heterogeneous-Nuclear Ribonucleoproteins
Replication Protein A
Processing
Nucleotides
DNA
Substrates
Flaps
Accessories
Eukaryotic Cells
DNA Replication
Immunoprecipitation
Ligation
Carrier Proteins
Okazaki fragments
RNA
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Interaction and Stimulation of Human FEN-1 Nuclease Activities by Heterogeneous Nuclear Ribonucleoprotein A1 in α-Segment Processing during Okazaki Fragment Maturation. / Chai, Qing; Zheng, Li; Zhou, Mian; Turchi, John; Shen, Binghui.

In: Biochemistry, Vol. 42, No. 51, 30.12.2003, p. 15045-15052.

Research output: Contribution to journalArticle

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abstract = "High-fidelity DNA replication depends on both accurate incorporation of nucleotides in the newly synthesized strand and the maturation of Okazaki fragments. In eukaryotic cells, the latter is accomplished by a series of coordinated actions of a set of structure-specific nucleases, which, with the assistance of accessory proteins, recognize branched RNA/DNA configurations. In the current model of Okazaki fragment maturation, displacement of a 27-nucleotide or longer flap is envisioned to attract replication protein A (RPA), which inhibits flap endonuclease-1 (FEN-1) but stimulates Dna2 nuclease for cleavage. Dna2 cleavage generates a short flap of 5-7 nucleotides, which resists binding by RPA and further cleavage by Dna2. FEN-1 then removes the remaining flap to produce a suitable substrate for ligation. However, FEN-1 is not efficient in cleaving the short flap, and we therefore set out to identify cellular factors that might regulate FEN-1 activity. Through co-immunoprecipitation experiments, we have isolated heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), which forms a direct complex with FEN-1 and stimulates its enzymatic activities. The stimulation by hnRNP A1 is most dramatic using DNA substrates with short flaps. With longer flap substrates the hnRNP A1 effect is more modest and is suppressed by the addition of RPA. A model is provided to explain the possible in vivo role of this interaction and activity in Okazaki fragment maturation.",
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