Distinct roles of two separable in vitro activities of yeast Mre11 in mitotic and meiotic recombination.
In Saccharomyces cerevisiae, Mre11 protein is involved in both double-strand DNA break (DSB) repair and meiotic DSB formation. Here, we report the correlation of nuclease and DNA-binding activities of Mre11 with its functions in DNA repair and meiotic DSB formation. Purified Mre11 bound to DNA efficiently and was shown to have Mn2+-dependent nuclease activities. A point mutation in the N-terminal phosphoesterase motif (Mre11D16A) resulted in the abolition of nuclease activities but had no significant effect on DNA binding. The wild-type level of nuclease activity was detected in a C-terminal truncated protein (Mre11DeltaC49), although it had reduced DNA-binding activity. Phenotypes of the corresponding mutations were also analyzed. The mre11D16A mutation conferred methyl methanesulfonate-sensitivity to mitotic cells and caused the accumulation of unprocessed meiotic DSBs. The mre11DeltaC49 mutant exhibited almost wild-type phenotypes in mitosis. However, in meiosis, no DSB formation could be detected and an aberrant chromatin configuration was observed at DSB sites in the mre11DeltaC49 mutant. These results indicate that Mre11 has two separable functional domains: the N-terminal nuclease domain required for DSB repair, and the C-terminal dsDNA-binding domain essential to its meiotic functions such as chromatin modification and DSB formation.


BACKGROUND
DNA binding/double-strand break repair/DSB formation/Mre11/nuclease
Furuse
M
M
Cellular and Molecular Biology Laboratory, The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-0198, Japan.
Nagase
Y
Y
Tsubouchi
H
H
Murakami-Murofushi
K
K
Shibata
T
T
Ohta
K
K
D017104:Bacteriophage M13 / Q000235:genetics; D002843:Chromatin / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008540:Meiosis / Q000235:genetics*; D008741:Methyl Methanesulfonate / Q000378:metabolism; D008938:Mitosis / Q000235:genetics*; D009154:Mutation; D010641:Phenotype; D011487:Protein Conformation; D011995:Recombination, Genetic / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D016615:Telomere / Q000235:genetics
D002843:Chromatin; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D008741:Methyl Methanesulfonate; D003851:Deoxyribonucleases; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C094191:MRE11 protein, S cerevisiae