A Role for USP7 in DNA Replication

Abstract

The minichromosome maintenance (MCM) complex, which plays multiple important roles in DNA replication, is loaded onto chromatin following mitosis, remains on chromatin until the completion of DNA synthesis, and then is unloaded by a poorly defined mechanism that involves the MCM binding protein (MCM-BP). Here we show that MCM-BP directly interacts with the ubiquitin-specific protease USP7, that this interaction occurs predominantly on chromatin, and that MCM-BP can tether USP7 to MCM proteins. Detailed biochemical and structure analyses of the USP7-MCM-BP interaction showed that the 155 PSTS 158 MCM-BP sequence mediates critical interactions with the TRAF domain binding pocket of USP7. Analysis of the effects of USP7 knockout on DNA replication revealed that lack of USP7 results in slowed progression through late S phase without globally affecting the fork rate or origin usage. Lack of USP7 also resulted in increased levels of MCM proteins on chromatin, and investigation of the cause of this increase revealed a defect in the dissociation of MCM proteins from chromatin in mid-to late S phase. This role of USP7 mirrors the previously described role for MCM-BP in MCM complex unloading and suggests that USP7 works with MCM-BP to unload MCM complexes from chromatin at the end of S phase.T he faithful replication of eukaryotic genomes relies on the complex interplay of multiple factors. Starting with the assembly of the prereplicative complex (pre-RC) at replication origins in G 1 , the coordinated loading of replication factors onto DNA and origin activation at the start of S phase result in the bidirectional progression of the replication machinery (replisome) to carry out DNA synthesis. The minichromosome maintenance (MCM) complex is a hexameric complex consisting of MCMs 2 to 7 (MCM2-7) and is an essential component of the pre-RC as well as the replisome (1). It is loaded onto DNA as double heterohexamers in G 1 phase through the concerted actions of Cdc6, Cdt1, and ORC (together, the pre-RC) (1). In eukaryotes, the MCM complex is loaded in an ϳ20-fold excess over the number of replication origins, with only a fraction being required for replication (2-7). At the start of S phase, phosphorylation events mediated by S-CDK (S-phase cyclin-dependent kinase) and DDK (Dbf4-dependent kinase) result in the association of the MCM complex with Cdc45 and the GINS complex (Psf1-3 and Sld5) forming an active "CMG" helicase (8-10). However, only ϳ10% of loaded MCM double hexamers form active CMG helicases, and the remaining "inactive" MCM complexes are thought to license dormant origins (11). During replication, these "inactive" MCM complexes pose a barrier to replication and must be removed ahead of the replication fork. While Pif1 family helicases have been implicated in this process (12-14), a mechanism for MCM complex unloading from dormant origins and from terminating replisomes at the end of S phase is not well characterized.Proteomics experiments performed with MCM proteins in human cells revealed a strong interaction w...

Structural Characterization of Interaction between Human Ubiquitin-specific Protease 7 and Immediate-Early Protein ICP0 of Herpes Simplex Virus-1

“…Plasmids containing recombinant proteins with the N-terminal His 6 tag were transformed into Escherichia coli BL21 (DE3) strain. Proteins were expressed in M9 minimal medium containing 15 (32). Sequencespecific resonance assignment of the UBL1 was done using ABACUS software (33,34).…”

“…Nearly complete UBL1 NMR resonance assignment was obtained with 93.3% of all expected chemical shifts assigned. NMR distance restraints were obtained from three-dimensional 15 N-and 13 C-edited NOESY-HSQC experiments (32). H-bond restraints were added on the basis of NOE analysis.…”

“…H-bond restraints were added on the basis of NOE analysis. Backbone dihedral and angle restraints were derived from the analysis of 15 N, 1 HN, 1 H␣, 13 C␣, 13 C␤, and 13 CO chemical shifts using TALOSϩ (35). Structures were calculated using CYANA (36).…”

“…The backbone resonance assignments of the 15 N/ 13 C-labeled deuterated UBL12 construct in its free and ICP0-bound forms were performed using two-dimensional 1 15 N-labeled UBL1 were performed at 25°C on 500 MHz ( 1 H) Agilent VNMRS spectrometer as described elsewhere (39,40). All experiments were performed on a C576S UBL1 mutant generated using the QuikChange kit (Agilent) to avoid slow cysteine-induced dimerization.…”

“…1A (19,24,25). USP7 complexes with several substrates have been previously characterized, including peptides derived from p53, Hdm2, HdmX, UbE2E1, MCM-BP, vlRF4, and EBNA1 (7,14,15,17,20,26). Remarkably, all these substrates share a common USP7-binding (P/A)XXS motif and interact with the same site located in the TRAF-like domain.…”