THE ROLE OF THE 70-KDA SUBUNIT OF HUMAN DNA POLYMERASE-ALPHA IN DNA-REPLICATION

DNA polymerase alpha is the only enzyme in eukaryotic cells capable of starting DNA chains de novo and is required for the initiation of SV4 0 DNA replication in vitro. We have cloned the 70 kDa subunit of human DNA polymerase alpha (hereafter referred to as the B subunit) and exp ressed it as a fusion protein in bacteria: The purified fusion protein forms a stable complex with SV40 T antigen, both in solution and when T antigen is bound to the SV40 origin of DNA replication. Analysis of mutant forms of the B subunit indicates that the N-terminal 240 amino acids are sufficient to mediate complex formation. The B subunit fusi on protein promotes formation of a complex containing T antigen and th e catalytic subunit (subunit A) of DNA polymerase alpha, suggesting th at it serves to tether the two proteins. These physical interactions a re functionally significant, since the ability of T antigen to stimula te the activity of the catalytic subunit of DNA polymerase alpha is hi ghly dependent upon the B subunit. We suggest that the interactions me diated by the B subunit play an important role in SV40 DNA replication by promoting DNA chain initiation at the origin and/or facilitating t he subsequent priming and synthesis of DNA chains on the lagging stran d template. The protein may play similar roles in cellular DNA replica tion.