The mechanism of Mus81-Mms4 cleavage site selection distinguishes it from the homologous endonuclease Rad1-Rad10.
Mus81-Mms4 and Rad1-Rad10 are homologous structure-specific endonucleases that cleave 3' branches from distinct substrates and are required for replication fork stability and nucleotide excision repair, respectively, in the yeast Saccharomyces cerevisiae. We explored the basis of this biochemical and genetic specificity. The Mus81-Mms4 cleavage site, a nick 5 nucleotides (nt) 5' of the flap, is determined not by the branch point, like Rad1-Rad10, but by the 5' end of the DNA strand at the flap junction. As a result, the endonucleases show inverse substrate specificity; substrates lacking a 5' end within 4 nt of the flap are cleaved poorly by Mus81-Mms4 but are cleaved well by Rad1-10. Genetically, we show that both mus81 and sgs1 mutants are sensitive to camptothecin-induced DNA damage. Further, mus81 sgs1 synthetic lethality requires homologous recombination, as does suppression of mutant phenotypes by RusA expression. These data are most easily explained by a model in which the in vivo substrate of Mus81-Mms4 and Sgs1-Top3 is a 3' flap recombination intermediate downstream of replication fork collapse.
Bastin-Shanower
Suzanne A
SA
Department of Molecular Biology and Biochemistry, CABM, Rutgers University, 679 Hoes Lane, Piscataway, NJ 08854, USA.
Fricke
William M
WM
Mullen
Janet R
JR
Brill
Steven J
SJ
D001483:Base Sequence; D004249:DNA Damage; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004720:Endonucleases / Q000378:metabolism*; D045585:Flap Endonucleases; D005656:Fungal Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013379:Substrate Specificity; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D014466:Ultraviolet Rays
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D004720:Endonucleases; D045585:Flap Endonucleases; C411624:MUS81 protein, S cerevisiae; C069266:RAD1 protein, S cerevisiae; C112337:MMS4 protein, S cerevisiae; C064162:RAD10 protein, S cerevisiae; D015719:Single-Strand Specific DNA and RNA Endonucleases; D045643:DNA Repair Enzymes