Interaction of checkpoint proteins Hus1/Rad1/Rad9 with DNA base excision repair enzyme MutY homolog in fission yeast, Schizosaccharomyces pombe.
The DNA glycosylase MutY homolog (MYH) is responsible for removing adenines misincorporated opposite DNA strands containing guanine or 7,8-dihydro-8-oxoguanine by base excision repair thereby preventing G:C to T:A mutations. MYH has been shown to interact with the proliferating cell nuclear antigen (PCNA) in both human and fission yeast Schizosaccharomyces pombe systems. Here we show that S. pombe (Sp) MYH physically interacts with all subunits of the PCNA-like checkpoint protein heterotrimer, SpRad9/SpRad1/SpHus1, in yeast extracts and when the individual subunits are expressed in bacteria. The SpHus1 and SpPCNA binding sites are located in discrete regions of SpMYH. Immunoprecipitation assays reveal that the interaction between SpHus1 and SpMYH increases dramatically after hydrogen peroxide treatment, and this increase in the SpHus1-SpMYH interaction correlates with the presence of SpHus1 phosphorylation. In contrast, the interaction between SpPCNA and SpMYH after hydrogen peroxide treatment remains nearly unchanged. SpMYH associates with SpHus1 in a complex of approximately 450 kDa, the reported native molecular mass of the SpRad9/SpRad1/SpHus1-MYC complex. A larger portion of SpMYH shifts to the 150-500-kDa regions after hydrogen peroxide treatment in comparison with untreated extracts. SpHus1 phosphorylation is substantially reduced in SpMYH Delta cells after hydrogen peroxide treatment. These data suggest that MYH may act as an adaptor to recruit checkpoint proteins to the DNA lesions.
Chang
Dau-Yin
DY
Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, Baltimore, Maryland 21201, USA.
Lu
A-Lien
AL
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D045647:DNA Glycosylases / Q000235:genetics; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D021122:Protein Subunits; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D021122:Protein Subunits; D029702:Schizosaccharomyces pombe Proteins; C106332:hus1 protein, S pombe; C069930:rad9 protein; D004720:Endonucleases; C499354:rad1 protein, S pombe; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase