The Exonucleolytic and Endonucleolytic Cleavage Activities of Human Exonuclease 1 Are Stimulated by an Interaction with the Carboxyl-terminal Region of the Werner Syndrome Protein

Abstract

Exonuclease 1 (EXO-1), a member of the RAD2 family of nucleases, has recently been proposed to function in the genetic pathways of DNA recombination, repair, and replication which are important for genome integrity. Although the role of EXO-1 is not well understood, its 5 to 3 -exonuclease and flap endonuclease activities may cleave intermediates that arise during DNA metabolism. In this study, we provide evidence that the Werner syndrome protein (WRN) physically interacts with human EXO-1 and dramatically stimulates both the exonucleolytic and endonucleolytic incision functions of EXO-1. The functional interaction between WRN and EXO-1 is mediated by a protein domain of WRN which interacts with flap endonuclease 1 (FEN-1). Thus, the genomic instability observed in WRN؊/؊ cells may be at least partially attributed to the lack of interactions between the WRN protein and human nucleases including EXO-1.Werner syndrome (WS) 1 is a hereditary premature aging disorder characterized by genome instability (1). WS cells display elevated chromosomal aberrations (2-4), replication defects (3, 5-8), abnormal recombination (9, 10), altered telomere dynamics (11), and hypersensitivity to DNA-damaging agents (12-16). The gene defective in WS, designated WRN (17), encodes a protein with DNA helicase (18,19) and exonuclease (20 -22) activities which presumably functions in DNA metabolism to preserve genome integrity. To understand the basis of WS, a number of groups have investigated WRN protein interactions (for review, see Ref. 23). The collective work indicates that WRN interacts functionally with proteins implicated in replication and DNA repair including replication protein A (RPA), p53, Ku, and polymerase ␦. These studies have enabled researchers to speculate about pathways of DNA metabolism in which WRN might participate; however, the precise functions of the WRN gene product in vivo are not well understood.Recently we reported a novel interaction of WRN protein with the human 5Ј-flap endonuclease/5Ј to 3Ј-exonuclease (FEN-1) (24), a DNA structure-specific nuclease implicated in DNA replication, recombination, and repair (25). WRN protein stimulates FEN-1 cleavage activity by a physical interaction with a COOH-terminal domain of the WRN protein (24). Another member of the RAD2 family to which FEN-1 belongs is human exonuclease 1 (EXO-1) (26). Like FEN-1, EXO-1 is a structure-specific endonuclease as well as an exonuclease (27, 28). EXO-1 has been implicated in a number of DNA metabolic pathways including mismatch correction, mitotic and meiotic recombination, and double strand break repair (29 -34). A role for EXO-1 in Okazaki fragment processing during DNA replication has been suggested based on its structure-specific endonuclease and RNase H activities that are similar to those of FEN-1 (30). Functional overlap between EXO-1 and FEN-1 (Rad27 in Saccharomyces cerevisiae) has been proposed from observations in yeast that exoI: rad27 double mutants are inviable (35) and that overexpression of yeast EXO-1 or human EXO-1 co...

Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair

“…WRN was shown to stimulate both the flap endonuclease activity of hExo1 and removal of the 5Ј-terminal nucleotide at a nick (30). Stimulation of the flap endonuclease activity of both Exo1 and FEN-1 required the C-terminal domain (residues 949-1432) of WRN (30,36). Interestingly, the C-terminal domains of WRN and BLM share 19% similarity and 12% identity (36).…”

“…This characteristic eliminates the possibility that the observed enhancement of hExo1 activity requires BLM-catalyzed DNA unwinding. Interestingly, the ability of a human RecQ family helicase to stimulate the activity of another protein without requiring ATP is not without precedent: BLM stimulates FEN-1 (35), and WRN stimulates hExo1, FEN-1, and translesion DNA polymerases via direct interactions that do not require ATP-driven unwinding (30,36,37). WRN was shown to stimulate both the flap endonuclease activity of hExo1 and removal of the 5Ј-terminal nucleotide at a nick (30).…”

“…Interestingly, the ability of a human RecQ family helicase to stimulate the activity of another protein without requiring ATP is not without precedent: BLM stimulates FEN-1 (35), and WRN stimulates hExo1, FEN-1, and translesion DNA polymerases via direct interactions that do not require ATP-driven unwinding (30,36,37). WRN was shown to stimulate both the flap endonuclease activity of hExo1 and removal of the 5Ј-terminal nucleotide at a nick (30). Stimulation of the flap endonuclease activity of both Exo1 and FEN-1 required the C-terminal domain (residues 949-1432) of WRN (30,36).…”

“…The interaction of BLM and hRad51 (42) can explain the species specificity that we observed for hRad51 in DNA pairing and offers the testable hypothesis that BLM also recruits hRad51 to the resected DNA through its shared interaction with hExo1. Given that the N-and C-terminal domains of BLM can independently interact with hRad51 (42) and that the C-terminal domain of BLM likely interacts with hExo1 (30,36), such a mutual recruitment scheme has some molecular support. The nucleoprotein filament then searches for DNA sequence homology and mediates DNA strand invasion and exchange (step IV).…”

The role of DNA exonucleases in protecting genome stability and their impact on ageing

“…(Note that WRN also binds to telomere proteins POT1 and TRF2 (Opresko et al 2002(Opresko et al , 2005). Interestingly, WRN also interacts physically with and stimulates EXO1 (Aggarwal et al 2010;Sharma et al 2003), a nuclease that is implicated both in Okazaki fragment processing and in telomere processing. This interaction may be important for either WRN or EXO1, or both, to process stalled or regressed replication forks and to maintain the T and D loops at telomeres.…”