Abstract
Human exonuclease 1 (EXO1) is involved in multiple DNA metabolism processes, including DNA repair and replication. Most of the fundamental roles of EXO1 have been described in yeast. Here, we report a biochemical characterization of human full-length EXO1. Prior to assay EXO1 on different DNA flap structures, we determined factors essential for the thermodynamic stability of EXO1. We show that enzymatic activity and stability of EXO1 on DNA is modulated by temperature. By characterization of EXO1 flap activity using various DNA flap substrates, we show that EXO1 has a strong capacity for degrading double stranded DNA and has a modest endonuclease or 5' flap activity. Furthermore, we report novel mechanistic insights into the processing of flap structures, showing that EXO1 preferentially cleaves one nucleotide inwards in a double stranded region of a forked and nicked DNA flap substrates, suggesting a possible role of EXO1in strand displacement.
Highlights
Human exonuclease 1 (EXO1) has been implicated in many different DNA metabolic processes, including DNA mismatch repair (MMR), micro-mediated end-joining, homologous recombination (HR), and replication [1,2,3,4,5,6]
The importance of such studies has recently been demonstrated by biochemical studies that followed up on the MMR-deficient phenotype of the EXO1-Glu-109-Lys mouse
Our results suggest that the main process by which EXO1 regulates the endo activity is via tracking, whereas the process of threading contributes minor after the 5 blocking at the ssDNA to hydrolysing the 5 flap of 20 nt (Figures 6C and 6D)
Summary
Human exonuclease 1 (EXO1) has been implicated in many different DNA metabolic processes, including DNA mismatch repair (MMR), micro-mediated end-joining, homologous recombination (HR), and replication [1,2,3,4,5,6]. Human EXO1 is implicated in MMR and contain conserved binding domains interacting directly with MLH1 and MSH2 [5,6,12,13]. Binding domains to MLH1, and MSH2 indicated at the COOH-terminus of full-length EXO1. Full-length EXO1 has only been partially characterized and this has been limited to study its biochemical activity at various salt and pH conditions [32]. A mutated form of EXO1, EXO1-Glu-109-Lys, was biochemically characterized and reported to be compromised in nuclease activity; the mutated amino acid was not located within the conserved region [7,35,36]. We studied the specificity of the endonuclease activity of EXO1 by using specific DNA substrates and report novel mechanistic insights into the processing of flap structures. We show that EXO1 preferentially cleaves one nucleotide inwards in a double stranded region of forked and nicked DNA flap substrates, suggesting a possible role of EXO1 in strand displacement
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