Characterization of telomere-binding activity of replication factor C large subunit p140

The large subunit of RFC (RFC p140) has been suggested to be associated wit h the 3'-end of elongating DNA primer and to recruit proliferating cell nuc lear antigen (PCNA) onto DNA polymerase delta. Previously, we isolated a cD NA clone encoding a DNA-binding domain of RFC p140 as a telomeric repeat (T TAGGG)n binding protein. This domain was shown to have a specific affinity for the 5'-phosphate ends of a telomere repeat sequence. In order to invest igate the structure and function of RFC p140, we constructed the full-lengt h recombinant RFC p140 as well as N- and/or C-terminal deleted mutants and analyzed their telomere-binding activities. South-Western blot and gel mobi lity shift analyses revealed that deletion of the N- but not the C-terminal region enhances recognition of the telomeric repeat sequence and 5'-phosph ate ends, suggesting the negative effect of the N-terminal region of the RF C p140 binding to the telomeric repeat. On the other hand, the C-terminal t runcated RFC inhibits the telomerase activity more than the N-terminal-dele ted and full-length RFC p140. The inhibitory effect of RFC p140 on telomera se activity is completely diminished by both terminal deletions. Thus, a ce rtain interaction of the N- and C-terminal regions is considered to be requ ired for RFC p140 to suppress telomerase activity. Taken together, these re sults suggest that both telomeric repeat-binding and telomerase inhibitory activities of RFC p140 are finely regulated by the intrinsic N- and C-termi nal regions. (C) 1999 Academic Press.