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Computational prediction and experimental validation of 5 DNA‐binding residues of human flap endonuclease 1 (hFEN1)
Author(s) -
Kanjanapangka Julie,
Finger Lawrence David,
Shen Binghui
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.878.2
Subject(s) - dna , protein data bank (rcsb pdb) , rnase h , okazaki fragments , microbiology and biotechnology , rnase p , dna clamp , genome instability , dna repair , biology , chemistry , biochemistry , dna damage , gene , rna , eukaryotic dna replication , reverse transcriptase
Human FEN1 is a structure‐specific DNA nuclease involved in removing 5′ RNA primers of Okazaki fragments and in long‐patch base excision repair. Defects in FEN1 lead to increased genomic instability. Identification of DNA‐binding residues will further elucidate the molecular mechanisms behind genome instability initiation and facilitate cancer therapy linked to hFEN1 mutations. No crystal structure data is available of hFEN1 actively bound to DNA; however, a structure of an hFEN1 homolog, T4 phage RNase H in its active form (pdb 2ihn), is available as well as hFEN1 complexed with PCNA in the inactive form (pdb 1ul1). The 2ihn pdb structure shows 31 DNA‐interacting residues. Computational structural alignment of the two X‐ray crystal structures has revealed 5 residues within the 31 subset of RNase H that have a seemingly structurally conserved equivalent in hFEN1; these are K74, L29, V30, F53, and H174. These conserved equivalents in hFEN1 may therefore interact with DNA. To validate our predictions, we use HPLC to measure specific activity levels of hFEN1 mutant proteins versus the wildtype using fluorescein‐labeled DNA substrate. We found that the specific activities of the mutants are impaired relative to the wildtype. We will measure DNA binding activity of hFEN1 mutants using gel shift with biotin‐labeled DNA substrate to validate if the results we observe are due to defects in DNA substrate binding. This work is funded by NIH R01CA076734: Fen1 in genome stability and cancer.