Characterization of the dimeric CMG/pre-initiation complex and its transition into DNA replication forks.

Characterization of the dimeric CMG/pre-initiation complex and its transition into DNA replication forks.
Cell Mol Life Sci. 2019 Nov 15;:
Authors: Liu L, Zhang Y, Zhang J, Wang JH, Cao Q, Li Z, Campbell JL, Dong MQ, Lou H
Abstract
The pre-initiation complex (pre-IC) has been proposed for two decades as an intermediate right before the maturation of the eukaryotic DNA replication fork. However, its existence and biochemical nature remain enigmatic. Here, through combining several enrichment strategies, we are able to isolate an endogenous dimeric CMG-containing complex (designated as d-CMG) distinct from traditional single CMG (s-CMG) and in vitro reconstituted dimeric CMG. D-CMG is assembled upon entry into the S phase and shortly matures into s-CMG/replisome, leading to the fact that only ~ 5% of the total CMG-containing complexes can be detected as d-CMG in vivo. Mass spectra reveal that RPA and DNA Pol α/primase co-purify with s-CMG, but not with d-CMG. Consistently, the former fraction is able to catalyze DNA unwinding and de novo synthesis, while the latter catalyzes neither. The two CMGs in d-CMG display flexibly orientated conformations under an electronic microscope. When DNA Pol α-primase is inactivated, d-CMG % rose up to 29%, indicating an incomplete pre-IC/fork transition. These findings reveal biochemical properties of the d-CMG/pre-IC and provide in vivo evidence to support the pre-IC/fork transition as a bona f...

Source: Cellular and Molecular Life Sciences : CMLS - November 14, 2019 Category: Cytology Authors: Liu L, Zhang Y, Zhang J, Wang JH, Cao Q, Li Z, Campbell JL, Dong MQ, Lou H Tags: Cell Mol Life Sci Source Type: research