Physical and functional interaction between human oxidized base-specific DNA glycosylase NEIL1 and flap endonuclease 1.
The S phase-specific activation of NEIL1 and not of the other DNA glycosylases responsible for repairing oxidatively damaged bases in mammalian genomes and the activation of NEIL1 by proliferating cell nuclear antigen (PCNA) suggested preferential action by NEIL1 in oxidized base repair during DNA replication. Here we show that NEIL1 interacts with flap endonuclease 1 (FEN-1), an essential component of the DNA replication. FEN-1 is present in the NEIL1 immunocomplex isolated from human cell extracts, and the two proteins colocalize in the nucleus. FEN-1 stimulates the activity of NEIL1 in vitro in excising 5-hydroxyuracil from duplex, bubble, forked, and single-stranded DNA substrates by up to 5-fold. The disordered region near the C terminus of NEIL1, which is dispensable for activity, is necessary and sufficient for high affinity binding to FEN-1 (K(D) approximately = 0.2 microm). The interacting interface of FEN-1 is localized in its disordered C-terminal region uniquely present in mammalian orthologs. Fine structure mapping identified several Lys and Arg residues in this region that form salt bridges with Asp and Glu residues in NEIL1. NEIL1 was previously shown to initiate single nucleotide excision repair, which does not require FEN-1 or PCNA. The present study shows that NEIL1 could also participate in strand displacement repair synthesis (long patch repair (LP-BER)) mediated by FEN-1 and stimulated by PCNA. Interaction between NEIL1 and FEN-1 is essential for efficient NEIL1-initiated LP-BER. These studies strongly implicate NEIL1 in a distinct subpathway of LP-BER in replicating genomes.
Hegde
Muralidhar L
ML
Department of Biochemistry and Molecular Biology, University of Texas, Medical Branch, Galveston, Texas 77555, USA.
Theriot
Corey A
CA
Das
Aditi
A
Hegde
Pavana M
PM
Guo
Zhigang
Z
Gary
Ronald K
RK
Hazra
Tapas K
TK
Shen
Binghui
B
Mitra
Sankar
S
D001665:Binding Sites / Q000502:physiology; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000201:enzymology / Q000235:genetics; D004249:DNA Damage / Q000502:physiology*; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D004261:DNA Replication / Q000502:physiology*; D004789:Enzyme Activation; D045585:Flap Endonucleases / Q000235:genetics / Q000378:metabolism*; D015894:Genome, Human / Q000502:physiology*; D006801:Humans; D010084:Oxidation-Reduction; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D016196:S Phase / Q000502:physiology
D018809:Proliferating Cell Nuclear Antigen; D045585:Flap Endonucleases; C468317:FEN1 protein, human; D045647:DNA Glycosylases; C473634:NEIL1 protein, human