Abstract

DNA polymerase ε from HeLa cells has been purified as a heterodimer of a 261 kDa catalytic subunit and a tightly associated smaller polypeptide, the B-subunit. The cDNAs encoding the B-subunits of both human and mouse Pol ε were cloned and shown to encode proteins with a predicted molecular weight of 59 kDa. These subunits are 90 % identical and share 22 % identity with the 80 kDa B-subunit of Saccharomyces cerevisiae Pol ε. The gene for the human Pol ε B-subunit was localized to chromosome 14q21-q22 by fluorescence in situ hybridization.

Primary structure analysis of the Pol ε B-subunits demonstrated that they are similar to the B-subunits of Pol α, Pol δ and archaeal DNA polymerases, and comprise a novel protein family of DNA polymerase associated-B-subunits. The family members have 12 conserved motifs distributed in the C-terminal parts, which apparently form crucial structural and functional sites. Secondary structure predictions indicate that the B-subunits share a similar fold, and phylogenetic analysis demonstrated that the B-subunits of Pol α and ε form one subfamily, while the B-subunits of Pol δ and the archaeal proteins form a second subfamily. The corresponding eukaryotic and archaeal catalytic subunits are not related, but all have the characteristics of replicative DNA polymerases. This indicates that the B-subunits of replicative DNA polymerases from archaea to eukaryotes belong to the same protein family and perform similar functions.

In S. cerevisiae, Pol ε associates with the checkpoint protein Dpb11. In this study, a human protein, TopBP1, with structural similarity to the budding yeast Dpb11, fission yeast Cut5 and the breast cancer susceptibility gene product Brca1 was identified. The human TOPBP1 gene localizes to chromosome 3q21-q23 and encodes a phosphoprotein of 180 kDa. TopBP1 has eight BRCT domains and is also closely related to the recently identified Drosophila melanogaster Mus101. TopBP1 expression is induced at the G1/S boundary and it performs an important role in DNA replication, as evidenced by inhibition of DNA synthesis by TopBP1 antiserum in isolated nuclei. TopBP1 also associates with Pol ε and localizes, together with Brca1 to distinct foci in S-phase, but not to sites of ongoing DNA replication. Inhibition of DNA replication leads to re-localization of TopBP1 and Brca1 to stalled replication forks. DNA damage induces formation of distinct TopBP1 foci that co-localize with Brca1 in S-phase, but not in G1-phase. The role of TopBP1 in the DNA damage response is also supported by the interaction between TopBP1 and the human checkpoint protein hRad9. These results implicate TopBP1 in replication and checkpoint functions.