ATP binding and hydrolysis by Mcm2 regulate DNA binding by Mcm complexes.
The essential minichromosome maintenance (Mcm) proteins Mcm2 through Mcm7 likely comprise the replicative helicase in eukaryotes. In addition to Mcm2-7, other subcomplexes, including one comprising Mcm4, Mcm6, and Mcm7, unwind DNA. Using Mcm4/6/7 as a tool, we reveal a role for nucleotide binding by Saccharomyces cerevisiae Mcm2 in modulating DNA binding by Mcm complexes. Previous studies have shown that Mcm2 inhibits DNA unwinding by Mcm4/6/7. Here, we show that interaction of Mcm2 and Mcm4/6/7 is not sufficient for inhibition; rather, Mcm2 requires nucleotides for its regulatory role. An Mcm2 mutant that is defective for ATP hydrolysis (K549A), as well as ATP analogues, was used to show that ADP binding by Mcm2 is required to inhibit DNA binding and unwinding by Mcm4/6/7. This Mcm2-mediated regulation of Mcm4/6/7 is independent of Mcm3/5. Furthermore, the importance of ATP hydrolysis by Mcm2 to the regulation of the native complex was apparent from the altered DNA binding properties of Mcm2(KA)-7. Moreover, together with the finding that Mcm2(K549A) does not support yeast viability, these results indicate that the nucleotide-bound state of Mcm2 is critical in regulating the activities of Mcm4/6/7 and Mcm2-7 complexes.
Stead
Brent E
BE
Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, Ontario, Canada.
Sorbara
Catherine D
CD
Brandl
Christopher J
CJ
Davey
Megan J
MJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone; D004271:DNA, Fungal / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D046912:Multiprotein Complexes / Q000378:metabolism*; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D002868:Chromosomal Proteins, Non-Histone; D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D005656:Fungal Proteins; D046912:Multiprotein Complexes; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; C069199:MCM2 protein, S cerevisiae