PHOSPHORYLATION OF THE P34 SUBUNIT OF HUMAN SINGLE-STRANDED-DNA-BINDING PROTEIN IN CYCLIN-A-ACTIVATED G(1) EXTRACTS IS CATALYZED BY CDK CYCLIN-A COMPLEX AND DNA-DEPENDENT PROTEIN-KINASE

The human single-stranded-DNA-binding protein (HSSB, also called RP-A) is a trimeric complex (p70, p34, and p14) required for multiple functions in DNA transactions. We report here that the p34 subunit of HSSB was hyperphosphorylated by kinase activities present in G(1) extract (obtained from HeLa cells in G(1) phase) preincubated with human cyclin A. This hyperphosphorylated HSSB product included at least four species of p34 that migrated more slowly through denaturing polyacrylamide gels than the hypophosphorylated form. Fractionation of cyclin A-activated G(1) extract identified two kinases involved in the hyperphosphorylation of HSSB p34: cdk-cyclin A complex and DNA-dependent p350 protein kinase (DNA-PK). Kinetic analysis revealed that in cyclin A-activated G(1) extract, p34 was first phosphorylated by cdk-cyclin A prior to the action of DNA-PK. Addition of p21(cip1), a specific inhibitor of cdk-cyclin A but not DNA-PK, nearly abolished the hyperphosphorylation of HSSB p34 in G(1) extract preincubated with cyclin A. This suggests a requirement of the cdk-cyclin A activity for the phosphorylation of p34 by DNA-PK in G(1) extract.