In this study we have investigated the molecular basis and physiological relevance of the constitutive association between pol α and primase, two essential components of the eukaryotic replisome. We have identified a short sequence motif in the C-terminal tail of pol α that is conserved from yeast to humans and is critical for the interaction with primase. In agreement with the in vitro data, a version of Pol1 bearing the F1463A mutation is unable to associate with the primase in yeast cells, and the pol1-F1463A strain becomes dependent on the S phase checkpoint for survival. The functional importance of the primase binding motif of pol α is further highlighted by the observation that removal of the last 16 amino acids of Pol1 blocks yeast cell proliferation. Taken together, our data indicate that primase recruitment to the eukaryotic replisome is critically dependent on a short conserved motif in the C-terminal tail of the catalytic subunit of pol α (E).
Our findings agree with and extend earlier observations that the C-terminal 200 amino acids of pol α mediate interaction with primase and B subunit (15
). We note that a previous study showed that removal of the last 67 amino acids from a recombinant form of human pol α abolished interaction with the B subunit (16
). Association of the truncated protein with primase was greatly reduced but not completely abolished (19
), and we cannot exclude the possibility that the critical interaction of primase with the C-terminal tail of pol α might be fortified in the case of the human complex by additional contacts within the C-terminal region of the catalytic subunit.
The important role of short linear motifs in mediating protein-protein interactions is increasingly being recognized (24
). The primase binding sequence of pol α displays the salient features observed in this class of protein-protein interactions. It is natively unstructured, as it was disordered in the crystal structure of the yeast CTD-B subunit complex. Its conserved positions are enriched for hydrophobic and especially aromatic residues. Finally, primase binding relies critically on “hot spot” residues that make a dominant contribution to the binding energy (24
). In fact, primase binding is effectively abrogated by the point mutation of an invariant phenylalanine, F1463A in Pol1 or Phe1455
in human pol α, as determined by GST pulldown, fluorescence anisotropy, and immunoprecipitation from yeast cells.
Although the pol1-F1463A
strain is viable, its dependence for survival on the checkpoint kinases Mec1 and Rad53 (data not shown) implies the existence of a subtle replication defect. The difference in the phenotype of the pol1-F1463A
and pol1 1–1452
alleles is in intriguing contrast with their similar biochemical effects. The inviability of the pol1 1–1452
allele might reflect additional interactions that are mediated by the C-terminal tail of pol1. Alternatively, pol1-F1463A might retain a minimal level of primase binding that is still able to support a viable level of primase incorporation in the replisome, even though this is not detectable experimentally. We note that the mutant pol1 proteins showed reduced levels of association with other core components of the replisome. Previous work had indicated that Pol1 incorporation into the replisome is critically dependent on Ctf4, via an interaction mediated by the N-terminal region of Pol1 (10
). It is plausible that, in addition to the known Pol1-Ctf4 interaction, stable integration of the pol α-primase complex into the replisome might require additional contacts with replisome components that are mediated by primase.
Our findings highlight the importance of tethering primase to the eukaryotic replisome during chromosome replication. The limited size of the primase-binding epitope of pol α suggests that it might be possible to design small molecule inhibitors that disrupt the pol α-primase interface. As loss of the interaction of primase with pol α makes cells dependent upon the S phase checkpoint response, such inhibitors might have therapeutic potential in the treatment of human cancers with inherent defects in chromosome replication.