Biochemical activities associated with mouse Mcm2 protein.

Yukio Ishimi,Yuki Komamura-Kohno,Ken-ichi Arai,Hisao Masai

doi:10.1074/jbc.m106861200

Abstract

Mcm2, a member of the Mcm2-7 protein family essential for the initiation of DNA replication, has several biochemical activities including the ability to inhibit the Mcm4,6,7 helicase. In this study, we characterized the activities associated with Mcm2 and determined the region required for them. It was found that Mcm2 deleted at an amino-terminal portion is able to bind to an Mcm4,6,7 hexameric complex and to inhibit its DNA helicase activity. The same deletion mutant of Mcm2 and the carboxyl-terminal half of Mcm2 were both able to bind to Mcm4, suggesting that the carboxyl-half of Mcm2 binds to Mcm4 to disassemble the Mcm4,6,7 hexamer. Phosphorylation of Mcm2,4,6,7 complexes with Cdc7 kinase showed that the amino-terminal region of Mcm2 is required for the phosphorylation, and it contains major Cdc7-mediated phosphorylation sites. We also found that Mcm2 itself can assemble a nucleosome-like structure in vitro in the presence of H3/H4 histones. The amino-terminal region of Mcm2 was required for the activity where a histone-binding domain is located. Finally, we identified a region required for the nuclear localization of Mcm2. The function of Mcm2 is discussed based on these biochemical characteristics.

Highlights

All the Mcm2–7 proteins play an essential and distinct role in eukaryotic DNA replication [1,2,3]
Mcm2 and Mcm3/5 can inhibit the DNA helicase activity by disassembling the Mcm4,6,7 hexamer into an Mcm2,4,6,7 complex or an Mcm3,4,5,6,7 complex, respectively [13, 17, 18]. These results indicate that the assembly of Mcm4,6,7 proteins into a hexamer is crucial for the DNA helicase activity and suggest that Mcm2, -3, and -5 proteins play a role in regulating the Mcm4,6,7 helicase activity
We reported that Mcm2 can inhibit the DNA helicase activity of the Mcm4,6,7 complex [17]

Summary

Introduction

All the Mcm2–7 proteins play an essential and distinct role in eukaryotic DNA replication [1,2,3]. Mcm2 deleted at the amino-terminal end [1–162] showed inhibition of DNA helicase activity similar to the full-sized Mcm2 protein [17]. To map the region in Mcm2 that is required for the phosphorylation with the Cdc7/ASK kinase, we constructed four Mcm2 proteins deleted at the amino-terminal region and purified Mcm2,4,6,7 complexes containing these Mcm2 (Fig. 4A).

Results

Conclusion