Interaction interface of human flap endonuclease-1 with its DNA substrates.

Brian R. Chapados,Binghui Shen,John A. Tainer,Mark Sherman,Junzhuan Qiu,Ren Liu

doi:10.1074/jbc.m401464200

Abstract

Flap endonuclease-1 or FEN-1 is a structure-specific and multifunctional nuclease critical for DNA replication, repair, and recombination; however, its interaction with DNA substrates has not been fully understood. In the current study, we have defined the borders of the interaction between the FEN-1 protein and its DNA substrates and identified six clusters of conserved positively charged amino acid residues, which are in direct contact with DNA substrate. To map further the corresponding interactions between FEN-1 residues and DNA substrates, we performed biochemical assays employing a series of flap DNA substrates lacking some structural components and a series of binding-deficient point mutants of FEN-1. It was revealed that Arg(47), Arg(70), and Lys(326)-Arg(327) of FEN-1 interact with the upstream duplex of DNA substrates, whereas Lys(244)-Arg(245) interact with the downstream duplex. This result indicates the orientation of the FEN-1-DNA interaction. Moreover, Arg(70) and Arg(47) were determined to interact with the sites around the 2nd nucleotide (Arg(70)) or the 5th/6th nucleotide (Arg(47)) of the template strand in the upstream duplex portion counting from the nick point of the flap substrate. Together with previously published data and the crystallographic ainformation from the FEN-1.DNA complex that we published recently (Chapados, B. R., Hosfield, D. J., Han, S., Qiu, J., Yelent, B., Shen, B., Tainer, J. A. (2004) Cell 116, 39-50) we are able to propose a reasonable model for how the human FEN-1 protein interacts with its DNA substrates.

Highlights

Flap endonuclease-1 or FEN-1 is a structure-specific and multifunctional nuclease critical for DNA replication, repair, and recombination; its interaction with DNA substrates has not been fully understood
We defined the region of substrate DNA protected by FEN-1 binding and identified a number of positively charged amino acids critical for FEN-1 binding to DNA
The exonuclease III (exoIII) footprinting analysis indicates that FEN-1 protects 13 nucleotides of the downstream duplex and 20 nucleotides of the upstream duplex

Summary

EXPERIMENTAL PROCEDURES

The indicated amounts of FEN-1 were mixed with 80 nM labeled DNA in a final volume of 10 ␮l containing 50 mM Tris (pH 8.0), 10 mM NaCl, 5 mM EDTA, 10% glycerol, and 50 ␮g/ml bovine serum albumin. Human FEN-1 mutant R100A or D181A was used to bind 0.27-pmol flap substrates with 5Ј-32P end labeling of the flap strand or template strand. They were incubated in 50 mM Tris (pH 6.8), 20 mM NaCl, 15% glycerol, and 50 ␮g/ml bovine serum albumin with 2 mM MgCl2 and 100-fold excess cold double-stranded DNA competitor for 20 min. CD data were analyzed using the K2D program [37] for the calculation of the relative ratio of ␣-helices, ␤-sheets, and random coils

RESULTS

Interactive DNA region

DISCUSSION