Domain Architecture and Biochemical Characterization of Vertebrate Mcm10

Patrick D Robertson,Eric M Warren,Haijiang Zhang,David B Friedman,Jeffrey W Lary,James L Cole,Antonin V Tutter,Johannes C Walter,Ellen Fanning,Brandt F Eichman

doi:10.1074/jbc.m706267200

Abstract

Mcm10 plays a key role in initiation and elongation of eukaryotic chromosomal DNA replication. As a first step to better understand the structure and function of vertebrate Mcm10, we have determined the structural architecture of Xenopus laevis Mcm10 (xMcm10) and characterized each domain biochemically. Limited proteolytic digestion of the full-length protein revealed N-terminal-, internal (ID)-, and C-terminal (CTD)-structured domains. Analytical ultracentrifugation revealed that xMcm10 self-associates and that the N-terminal domain forms homodimeric assemblies. DNA binding activity of xMcm10 was mapped to the ID and CTD, each of which binds to single- and double-stranded DNA with low micromolar affinity. The structural integrity of xMcm10-ID and CTD is dependent on the presence of bound zinc, which was experimentally verified by atomic absorption spectroscopy and proteolysis protection assays. The ID and CTD also bind independently to the N-terminal 323 residues of the p180 subunit of DNA polymerase alpha-primase. We propose that the modularity of the protein architecture, with discrete domains for dimerization and for binding to DNA and DNA polymerase alpha-primase, provides an effective means for coordinating the biochemical activities of Mcm10 within the replisome.

Highlights

Pre-replicative complexes composed of the origin recognition complex, Cdc6, Cdt1, and the hexameric Mcm2–7 helicase are assembled in G1 and converted into active replication forks at the onset of S phase
XMcm10 Contains Three Structural Domains—In the current study experiments to characterize the domain architecture of vertebrate Mcm10 were carried out using the X. laevis ortholog because of previous investigations of the function of the protein using Xenopus egg extracts [6, 40]
The purified MBPxMcm10-His6 protein was subjected to limited proteolytic digestion by trypsin, chymotrypsin, and elastase, and the major proteolytic fragments were identified by MALDI-TOF MS and MALDI

Summary

EXPERIMENTAL PROCEDURES

Expression, and Purification of xMcm10—The cDNAs encoding full-length xMcm (FL, 1– 860) and deletion fragments 1–145, 1–230, 230 – 427, 427– 860, and 596 – 860 were PCR-amplified from a previously described plasmid encoding a GST-xMcm fusion [6]. The cleaved proteins were further purified by cation exchange (fragments 230 – 427, 427– 860, 596 – 860) or anion exchange (1–145 and 1–230) chromatography followed by gel filtration on a SuperdexTM 200 preparative column (GE Healthcare) that had been equilibrated with S-200 buffer 2– 8 pmol of purified xMcm or 0.6 –2.4 pmol of purified polymerase ␣-primase were incubated at 37 °C for 40 min with 1.0 ␮M dT50, 25 ␮Ci of [␣-32P]ATP, and 0.1 mM ATP in a 10 ␮M reaction containing 40 mM Tris-HCl, pH 7.4, 10 mM magnesium acetate, 1 mM dithiothreitol, and 100 ␮g/ml bovine serum albumin.

RESULTS

ND ND

DISCUSSION