Comprehensive Mapping of the C-Terminus of Flap Endonuclease-1 Reveals Distinct Interaction Sites for Five Proteins That Represent Different DNA Replication and Repair Pathways

Department of Radiation Biology, City of Hope National Medical Center and Beckman Research Institute, Duarte, CA 91010, USA.
Journal of Molecular Biology (Impact Factor: 4.33). 04/2008; 377(3):679-90. DOI: 10.1016/j.jmb.2007.10.074
Source: PubMed


Flap endonuclease-1 (FEN-1) is a multifunctional and structure-specific nuclease that plays a critical role in maintaining human genome stability through RNA primer removal, long-patch base excision repair, resolution of DNA secondary structures and stalled DNA replication forks, and apoptotic DNA fragmentation. How FEN-1 is involved in multiple pathways, of which some are seemingly contradictory, is of considerable interest. To date, at least 20 proteins are known to interact with FEN-1; some form distinct complexes that affect one or more FEN-1 activities presumably to direct FEN-1 to a particular DNA metabolic pathway. FEN-1 consists of a nuclease core domain and a C-terminal extension. While the core domain harbors the nuclease activity, the C-terminal extension may be important for protein-protein interactions. Here, we have truncated or mutated the C-terminus of FEN-1 to identify amino acid residues that are critical for interaction with five proteins representing roles in different DNA replication and repair pathways. We found with all five proteins that the C-terminus is important for binding and that each protein uses a subset of amino acid residues. Replacement of one or more residues with an alanine in many cases leads to the complete loss of interaction, which may consequently lead to severe biological defects in mammals.

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    • "Consistent with the role of a disordered region, each interactive partner of FEN1 requires a distinct combination of extended C-terminal residues for binding. For instance, the Q337, L340, D342, F343, F344, V346, S353 and S352 residues within and next to the PIP box of FEN1 are essential for the FEN1/PCNA interaction, whereas the K366, K367, K375, R378 and K380 residues are required for the FEN1/WRN interaction (88). On the other hand, the lysine residues, including K375 and K380, of FEN1 have been shown to be acetylated by p300 (14). "
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    ABSTRACT: Flap endonuclease-1 (FEN1) is a member of the Rad2 structure-specific nuclease family. FEN1 possesses FEN, 5'-exonuclease and gap-endonuclease activities. The multiple nuclease activities of FEN1 allow it to participate in numerous DNA metabolic pathways, including Okazaki fragment maturation, stalled replication fork rescue, telomere maintenance, long-patch base excision repair and apoptotic DNA fragmentation. Here, we summarize the distinct roles of the different nuclease activities of FEN1 in these pathways. Recent biochemical and genetic studies indicate that FEN1 interacts with more than 30 proteins and undergoes post-translational modifications. We discuss how FEN1 is regulated via these mechanisms. Moreover, FEN1 interacts with five distinct groups of DNA metabolic proteins, allowing the nuclease to be recruited to a specific DNA metabolic complex, such as the DNA replication machinery for RNA primer removal or the DNA degradosome for apoptotic DNA fragmentation. Some FEN1 interaction partners also stimulate FEN1 nuclease activities to further ensure efficient action in processing of different DNA structures. Post-translational modifications, on the other hand, may be critical to regulate protein-protein interactions and cellular localizations of FEN1. Lastly, we also review the biological significance of FEN1 as a tumor suppressor, with an emphasis on studies of human mutations and mouse models.
    Nucleic Acids Research 10/2010; 39(3):781-94. DOI:10.1093/nar/gkq884 · 9.11 Impact Factor
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    • "FEN1 is a multifunctional protein and more than 20 proteins are known to interact with it. Recently, deletion and alanine substitution mutagenesis of FEN1 was used to map sites where PCNA, WRN, APE1, EndoG and the Rad9–Rad1–Hus1 complex interact with FEN1 (53). All the proteins tested interacted with the very C-terminus of FEN1. "
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    ABSTRACT: Human RECQL5 is a member of the RecQ helicase family which is implicated in genome maintenance. Five human members of the family have been identified; three of them, BLM, WRN and RECQL4 are associated with elevated cancer risk. RECQL1 and RECQL5 have not been linked to any human disorder yet; cells devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5beta, with the human flap endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5beta dramatically stimulates the rate of FEN1 cleavage of flap DNA substrates. Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes. This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage.
    Nucleic Acids Research 05/2010; 38(9):2904-16. DOI:10.1093/nar/gkp1217 · 9.11 Impact Factor
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    • " contacts throughout its length , including the core nuclease domain that remains in our rad27 - K325 * mutant ( Sakurai et al . 2005 ) . The multiple contacts also include segments of FEN - 1 that extend on either side its PIP box . In addition , a truncated mutant of the human flap endonuclease missing its PIP box is capable of binding to PCNA ( Guo et al . 2008 ) . Thus , all mutant forms that we have created should be capable of binding to PCNA with reduced affinity . Increasing the amount of PCNA should increase the amount of binding , and that may be partially responsible for suppression of the mutant phenotypes ."
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    ABSTRACT: During lagging-strand DNA replication in eukaryotic cells primers are removed from Okazaki fragments by the flap endonuclease and DNA ligase I joins nascent fragments. Both enzymes are brought to the replication fork by the sliding clamp proliferating cell nuclear antigen (PCNA). To understand the relationship among these three components, we have carried out a synthetic lethal screen with cdc9-p, a DNA ligase mutation with two substitutions (F43A/F44A) in its PCNA interaction domain. We recovered the flap endonuclease mutation rad27-K325* with a stop codon at residue 325. We created two additional rad27 alleles, rad27-A358* with a stop codon at residue 358 and rad27-pX8 with substitutions of all eight residues of the PCNA interaction domain. rad27-pX8 is temperature lethal and rad27-A358* grows slowly in combination with cdc9-p. Tests of mutation avoidance, DNA repair, and compatibility with DNA repair mutations showed that rad27-K325* confers severe phenotypes similar to rad27Delta, rad27-A358* confers mild phenotypes, and rad27-pX8 confers phenotypes intermediate between the other two alleles. High-copy expression of POL30 (PCNA) suppresses the canavanine mutation rate of all the rad27 alleles, including rad27Delta. These studies show the importance of the C terminus of the flap endonuclease in DNA replication and repair and, by virtue of the initial screen, show that this portion of the enzyme helps coordinate the entry of DNA ligase during Okazaki fragment maturation.
    Genetics 08/2009; 183(1):63-78. DOI:10.1534/genetics.109.103937 · 5.96 Impact Factor
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