Abstract
A dynamic multi-protein assembly known as the replisome is responsible for DNA synthesis in eukaryotic cells. In yeast, the hub protein Ctf4 bridges DNA helicase and DNA polymerase and recruits factors with roles in metabolic processes coupled to DNA replication. An important question in DNA replication is the extent to which the molecular architecture of the replisome is conserved between yeast and higher eukaryotes. Here, we describe the biochemical basis for the interaction of the human CTF4-orthologue AND-1 with DNA polymerase α (Pol α)/primase, the replicative polymerase that initiates DNA synthesis. AND-1 has maintained the trimeric structure of yeast Ctf4, driven by its conserved SepB domain. However, the primary interaction of AND-1 with Pol α/primase is mediated by its C-terminal HMG box, unique to mammalian AND-1, which binds the B subunit, at the same site targeted by the SV40 T-antigen for viral replication. In addition, we report a novel DNA-binding activity in AND-1, which might promote the correct positioning of Pol α/primase on the lagging-strand template at the replication fork. Our findings provide a biochemical basis for the specific interaction between two critical components of the human replisome, and indicate that important principles of replisome architecture have changed significantly in evolution.
Highlights
Duplication of the genome before cell division is performed by a multi-protein assembly known as the replisome [1,2]
Our experiments further showed that the interaction is mediated by the N-terminal regions of polymerase a (Pol a) and its B subunit, as no binding was observed in the case of the truncated Pol a/ primase
We have described an investigation into the interaction of AND-1, the human orthologue of the yeast replisome factor Ctf4, with DNA polymerase a/primase
Summary
Duplication of the genome before cell division is performed by a multi-protein assembly known as the replisome [1,2]. Integral to the replisome assembly are several non-enzymatic components that are important for efficient DNA replication, under normal conditions and in situations of replicative stress. One of the best characterized of these replisome components is the yeast Ctf (chromosome transmission fidelity 4), a homotrimeric hub that links the Cdc45-MCM-GINS (CMG) DNA helicase with DNA polymerase a (Pol a)/primase, and interacts with protein factors involved in various DNA metabolic processes, such as the helicase–nuclease Dna, the sister chromatid cohesion helicase Chl and the rDNA compaction protein Tof2 [3,4,5,6,7,8]. Ctf deficiency causes a pleiotropic phenotype, consisting of sensitivity to DNA damaging agents, faulty sister chromatid cohesion and alterations in the rDNA gene locus, which highlights its importance in maintaining genome stability during DNA replication [7,9,10,11,12]. Ctf orthologues have been identified in various eukaryotic organisms, including fission yeast (Mcl1) [13], Drosophila (Ctf4) [14] and humans (acidic and nucleoplasmic DNA-binding protein; AND-1) [15,16], suggesting that its functional role has been conserved throughout evolution
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