Identification and characterization of the human mus81-eme1 endonuclease
Alberto Ciccia1, Angelos Constantinou, Stephen C West
1Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
Abstract:
The faithful and complete replication of DNA is necessary for the maintenance of genome stability. It is known, however, that replication forks stall at lesions in the DNA template and need to be processed so that replication restart can occur. In fission yeast, the Mus81-Eme1 endonuclease complex (Mus81-Mms4 in Saccharomyces cerevisiae) has been implicated in the processing of aberrant replication intermediates. In this report, we identify the human homolog of the Schizosaccharomyces pombe EME1 gene and have purified the human Mus81-Eme1 heterodimer. We show that Mus81-Eme1 is an endonuclease that exhibits a high specificity for synthetic replication fork structures and 3'-flaps in vitro. The nuclease cleaves Holliday junctions inefficiently ( approximately 75-fold less than flap or fork structures), although cleavage can be increased 6-fold by the presence of homologous sequences previously shown to permit base pair "breathing." We conclude that human Mus81-Eme1 is a flap/fork endonuclease that is likely to play a role in the processing of stalled replication fork intermediates.
Insights
Human Mus81-Eme1 endonuclease processes stalled DNA replication forks. This flap/fork endonuclease is crucial for maintaining genome stability by resolving aberrant replication intermediates.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA replication is essential for genome stability.
- Replication forks can stall at DNA lesions, requiring processing for restart.
- The Mus81-Eme1 complex in yeast processes aberrant replication intermediates.
Purpose of the Study:
- Identify the human homolog of EME1.
- Purify and characterize the human Mus81-Eme1 heterodimer.
- Determine the enzymatic activity and substrate specificity of human Mus81-Eme1.
Main Methods:
- Gene identification and cloning.
- Protein purification of the human Mus81-Eme1 complex.
- In vitro enzymatic assays using synthetic DNA structures.
Main Results:
- Human Mus81-Eme1 was identified and purified.
- The enzyme is a flap/fork endonuclease with high specificity for these structures.
- It shows inefficient cleavage of Holliday junctions, enhanced by homologous sequences.
Conclusions:
- Human Mus81-Eme1 functions as a flap/fork endonuclease.
- This enzyme likely plays a critical role in processing stalled replication forks.
- Its activity contributes to maintaining genome stability.
