A conserved domain of the large subunit of replication factor C binds PCNA and acts like a dominant negative inhibitor of DNA replication in mammalian cells.

Abstract

Replication factor C (RF‐C), a complex of five polypeptides, is essential for cell‐free SV40 origin‐dependent DNA replication and viability in yeast. The cDNA encoding the large subunit of human RF‐C (RF‐Cp145) was cloned in a Southwestern screen. Using deletion mutants of RF‐Cp145 we have mapped the DNA binding domain of RF‐Cp145 to amino acid residues 369–480. This domain is conserved among both prokaryotic DNA ligases and eukaryotic poly(ADP‐ribose) polymerases and is absent in other subunits of RF‐C. The PCNA binding domain maps to amino acid residues 481–728 and is conserved in all five subunits of RF‐C. The PCNA binding domain of RF‐Cp145 inhibits several functions of RF‐C, such as: (i) in vitro DNA replication of SV40 origin‐containing DNA; (ii) RF‐C‐dependent loading of PCNA onto DNA; and (iii) RF‐C‐dependent DNA elongation. The PCNA binding domain of RF‐Cp145 localizes to the nucleus and inhibits DNA synthesis in transfected mammalian cells. In contrast, the DNA binding domain of RF‐Cp145 does not inhibit DNA synthesis in vitro or in vivo. We therefore conclude that amino acid residues 481–728 of human RF‐Cp145 are critical and act as a dominant negative mutant of RF‐C function in DNA replication in vivo.

Replication Factor C Clamp Loader Subunit Arrangement within the Circular Pentamer and Its Attachment Points to Proliferating Cell Nuclear Antigen

“…Interaction between RFC and PCNA-RFC1 is involved in a main connection to PCNA based on previous studies in the human system (25). The current study confirms this interaction in the yeast system and also demonstrates that the Tyr-404 and Phe-405 residues of RFC1 are involved in PCNA binding.…”

“…Possible explanations underlying the ATP dependence of the RFC(3/ 4)⅐PCNA complex and ATP-independent RFC(2/3)⅐PCNA complex are presented under "Discussion." We lack the RFC1 subunit in isolation or as a heterodimer with another RFC subunit, but we presume it also forms a major contact to PCNA based on studies of human RFC (25,32). The next few experiments take a different approach to assess the importance of RFC1 in RFC⅐PCNA complex formation.…”

“…Electron microscopy and atomic force microscopy studies of RFC are consistent with a circular arrangement of RFC subunits (9,24). The human RFC1 subunit (p140), like ␦, binds to PCNA (25), leading to the proposal that RFC1 may act to open PCNA just as the ␦ wrench opens ␤ (9, 18). RFC5, like ␦Ј, contains an SRC motif, and the putative ATP site deviates from the consensus sequence (GKKT instead of GKT) suggesting that if it can bind ATP, the ATP may not hydrolyze efficiently.…”

A Direct Interaction between Proliferating Cell Nuclear Antigen (PCNA) and Cdk2 Targets PCNA-interacting Proteins for Phosphorylation

“…The PCNA binding region of RF-Cp145 (RF-C B) and p21 were used as positive controls. The DNA binding region of RF-Cp145 (RF-C A) and GST were negative controls (21). Different salt concentrations for washing were used to test the salt dependence of the interaction between PCNA and Cdk2 (Fig.…”

Keeping chromatin quiet

“…Multiple interactions with PCNA may help to stabilize SMARCAD1's association with replication sites, similar to what has been observed for CAF1 and RFCp140. [64][65][66] Besides PCNA there may be additional mechanisms acting in parallel to target SMARCAD1 to ongoing replication. A precedent comes from DNMT1, whose interaction with PCNA is not essential for recruitment to replication foci while of a large number of ATP-dependent remodelers.…”