Human FEN1-PCNA Complex

 Maria Narvaez '14 and Hailey Schneider '14

Contents:

I. Introduction

Flap endonuclease-1 (FEN1) is an enzyme involved in the maintenance of genomic stability and replication. During replication of lagging strand DNA, FEN1 aids in removing DNA primers during Okazaki fragment maturation. FEN1 also plays an important role in long patch base excision repair. Studies have shown that the interaction of FEN1 with proliferating cell nuclear antigen (PCNA; the DNA sliding clamp) stimulates the activity of FEN1, leading to a 10- to 50-fold increase in its nuclease activity.

Mutations in human FEN1 impairing its endonuclease activity have been linked to cancer development, which makes sense considering its involvement in DNA replication and repair processes. 

II. General Structure

The human FEN1-PCNA complex is composed of 3 FEN1 molecules, bound to 1 PCNA trimer. The three PCNA subunits tightly associate to form a closed ring, with each subunit exhibiting symmetry. FEN1 and PCNA interact through beta-beta previous button is new!!!!!!!!!!!!!!! and hydrophobic interactions and these interactions keep the enzyme in an inactive locked-down orientation. It is possible that these interactions play a role in making rapid DNA-tracking possible by preserving the central hole of PCNA as it slides along the DNA.

Human FEN1 has two domains, the nucleus core domain and the C-terminal tail domain. The central groove of the core domain is formed by a group of beta sheets with two helical regions on both sides.


 

III. Activation

The human FEN1 active site, located at the central cleft, is formed by two clusters of conserved acidic residues that bind two metal ions. The first of these metal ion-binding sites is composed of Asp34, Asp86, Glu158, and Glu160. It is involved in the catalysis of nucleophilic attack of the phosphodiester bond of DNA. The second of the metal ion-binding sites is composed of Asp179, Asp181; and Asp233. This site is thought to be involved in DNA binding. The active site also involves the interaction of a conserved Tyr234 residue with Glu158, via a hydrogen bond, and with Asp181, via a water mediated contact.

The core domain of FEN1 is linked to the PCNA-binding tail by a short linker that acts as a hinge directing the FEN1 core domain towards the DNA substrate.

IV. DNA Binding

FEN1 interacts with the template DNA strand at two physically seperate regions, the H2TH:K+ ion and four basic residues: Arg239, Lys244, Arg245, and Lys267, which makes up the largest FEN1:DNA interface. The clamp region, which is believed to thread along the single strand DNA, contains many charged and hydrophobic residues. In addition to these phosphate interactions, the side chains of Glu-181 and Arg-185, both emanating from the recognition helix directly contact the bases within the major groove of the DNA. Because of the way that the protein binds to the DNA, a kink of ~40 degrees occurs between nucleotide base pairs six and seven on each side of the dyad axis, 5'-TG-3' This sequence has been shown to favor DNA flexibility and bending in other systems as well. Because of this kink, an additional five ionic interactions and four hydrogen bonds are able to occur between the protein and the DNA strand. Examples of these new interactions occur between Lys-26, Lys-166, His-199 and the DNA sugar-phosphate backbone The DNA bend is integral to the mechanism of transcription activation. Not only does it place CAP in the proper orientation for interaction with RNA polymerase, but wrapping the DNA around the protein may result in direct contacts between upstream DNA and RNA polymerase. 

V. Complex Disruption

A double mutation involving Pro253 (and Lys254) in yeast has been shown to affect the stimulation of FEN1-cite this?, with Pro253 thought to be involved in coordinating two oxygen atoms in the backbone of Ala252 and Pro253 toward betaA of FEN1. andTranscription activation by CAP requires more than merely the binding of cAMP and binding and bending of DNA. CAP contains an "activating region" that has been proposed to participate in direct protein-protein interactions with RNA polymerase and/or other basal transcription factors. Specifically, amino acids 156, 158, 159, and 162 have been proposed to be critical for transcription activation by CAP. These amino acids are part of a surface loop composed of residues 152-166 Researchers have concluded that the third and final step in transcription activation is this direct protein-protein contact between amino acids 156-162 of CAP, and RNA polymerase.


VI. References

Tsutakawa, Susan E., Scott Classen, Brian R. Chapados, Andrew S. Arvai, L. David Finger, Grant Guenther, Christopher G. Tomlinson, Peter Thompson, Altaf H. Sarker, Binghui Shen, Priscilla K. Cooper, Jane A. Grasby, and John A. Tainer. 2011. Human Flap Endonuclease Structures, DNA Double-Base Flipping, and a Unified Understanding of the FEN1 Superfamily. Cell 145:198-211.

Sakurai, Shigeru, Ken Kitano, Hiroto Yamaguchi, Keisuke Fukuda, Makiyo Uchida, Eiko Ohtsuka, Hiroshi Morioka, and Toshio Hakoshima. 2005. Structral Basis for Recruitment of Human Flap Endonuclease 1 to PCNA. The EMBO Journal 24.4: 683-93.

Vaney, Marie Christine, Gary L. Gilliland, James G. Harman, Alan Peterkofsky, and Irene T. Weber. 1989. Crystal Structure of a cAMP-Independent Form of Catabolite Gene Activator Protein with Adenosine Substituted in One of Two cAMP-Binding Sites. Biochemistry 28:4568-4574.

Weber, Irene T., Gary L. Gilliland, James G. Harman, and Alan Peterkofsky. 1987. Crystal Structure of a Cyclic AMP-independent Mutant of Catabolite Activator Protein. The Journal of Biological Chemistry 262:5630-5636.

Zhou, Yuhong, Ziaoping Zhang, and Richard H. Ebright. 1993. Identification of the activating region of catabolite gene activator protein (CAP): Isolation and characterization of mutants of CAP specifically defective in transcription activation. Proceedings of the National Academy of Sciences of the United States of America 90:6081-6085.

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