Interactions of DNA with human DNA primase monitored with photoactivatablecross-linking agents: Implications for the role of the p58 subunit

Regulation of the p49-p58 primase complex during primer synthesis and the i nteraction of the primase subunits with DNA were examined. After primase sy nthesizes a primer that DNA polymerase alpha (pol alpha) can readily elonga te, further primase activity is negatively regulated. This occurs within bo th the context of the four-subunit pol alpha-primase complex and in the p49 -p58 primase complex, indicating that the newly generated primer-template s pecies need not interact with pol alpha to regulate further primase activit y, Photo-cross-linking of single-stranded DNA-primase complexes revealed th at whereas the isolated p49 and p58 subunits both reacted with DNA upon pho tolysis, only the p58 subunit reacted with the DNA when photolysis was perf ormed using the p49-p58 primase complex. After primer synthesis by the comp lex, p58 was again the only subunit that reacted with the DNA, These result s suggest a model for regulation of primer synthesis in which the newly syn thesized primer-template species binds to p58 and regulates further primer synthesis. Additionally, the ability of p58 to interact with primer-templat e species suggests that p58 mediates the transfer of primers from the prima se active site to pol alpha.