Structural basis for the interaction of a hexameric replicative helicase with the regulatory subunit of human DNA polymerase α-primase.
DNA polymerase α-primase (Pol-prim) plays an essential role in eukaryotic DNA replication, initiating synthesis of the leading strand and of each Okazaki fragment on the lagging strand. Pol-prim is composed of a primase heterodimer that synthesizes an RNA primer, a DNA polymerase subunit that extends the primer, and a regulatory B-subunit (p68) without apparent enzymatic activity. Pol-prim is thought to interact with eukaryotic replicative helicases, forming a dynamic multiprotein assembly that displays primosome activity. At least three subunits of Pol-prim interact physically with the hexameric replicative helicase SV40 large T antigen, constituting a simple primosome that is active in vitro. However, structural understanding of these interactions and their role in viral chromatin replication in vivo remains incomplete. Here, we report the detailed large T antigen-p68 interface, as revealed in a co-crystal structure and validated by site-directed mutagenesis, and we demonstrate its functional importance in activating the SV40 primosome in cell-free reactions with purified Pol-prim, as well as in monkey cells in vivo.
Department of Molecular and Computational Biology, University of Southern California, Los Angeles, California 90089, USA.
Bo
B
Zhou
Diana R
DR
Arnett
Xian
X
Yu
Aaron
A
Brewster
Gregory A
GA
Sowd
Charlies L
CL
Xie
Stefan
S
Vila
Dahai
D
Gai
Ellen
E
Fanning
Xiaojiang S
XS
Chen
D001483:Base Sequence; D015139:Blotting, Southern; D004256:DNA Polymerase I / Q000737:chemistry / Q000378:metabolism*; D019915:DNA Primase / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004261:DNA Replication; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D013539:Simian virus 40 / Q000235:genetics
D017931:DNA Primers; D019915:DNA Primase; C116932:DNA polymerase alpha-primase; D004256:DNA Polymerase I