Interactions of DNA with human DNA primase monitored with photoactivatable cross-linking agents: implications for the role of the p58 subunit.
Regulation of the p49-p58 primase complex during primer synthesis and the interaction of the primase subunits with DNA were examined. After primase synthesizes a primer that DNA polymerase alpha (pol alpha) can readily elongate, further primase activity is negatively regulated. This occurs within both the context of the four-subunit pol alpha-primase complex and in the p49-p58 primase complex, indicating that the newly generated primer-template species need not interact with pol alpha to regulate further primase activity. Photo-cross-linking of single-stranded DNA-primase complexes revealed that whereas the isolated p49 and p58 subunits both reacted with DNA upon photolysis, only the p58 subunit reacted with the DNA when photolysis was performed using the p49-p58 primase complex. After primer synthesis by the complex, p58 was again the only subunit that reacted with the DNA. These results suggest a model for regulation of primer synthesis in which the newly synthesized primer-template species binds to p58 and regulates further primer synthesis. Additionally, the ability of p58 to interact with primer-template species suggests that p58 mediates the transfer of primers from the primase active site to pol alpha.
Arezi
B
B
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
Kirk
B W
BW
Copeland
W C
WC
Kuchta
R D
RD
D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004247:DNA / Q000378:metabolism*; D019915:DNA Primase / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010777:Photochemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D004247:DNA; D019915:DNA Primase