Many proteins involved in DNA replication and repair undergo post-translational modifications such as phosphorylation and ubiquitylation. Proliferating cell nuclear antigen (PCNA; a homotrimeric protein that encircles double-stranded DNA to function as a sliding clamp for DNA polymerases) is monoubiquitylated by the RAD6-RAD18 complex and further polyubiquitylated by the RAD5-MMS2-UBC13 complex in response to various DNA-damaging agents. PCNA mono- and polyubiquitylation activate an error-prone translesion synthesis pathway and an error-free pathway of damage avoidance, respectively. Here we show that replication factor C (RFC; a heteropentameric protein complex that loads PCNA onto DNA) was also ubiquitylated in a RAD18-dependent manner in cells treated with alkylating agents or H(2)O(2). A mutant form of RFC2 with a D228A substitution (corresponding to a yeast Rfc4 mutation that reduces an interaction with replication protein A (RPA), a single-stranded DNA-binding protein) was heavily ubiquitylated in cells even in the absence of DNA damage. Furthermore RFC2 was ubiquitylated by the RAD6-RAD18 complex in vitro, and its modification was inhibited in the presence of RPA. The inhibitory effect of RPA on RFC2 ubiquitylation was relatively specific because RAD6-RAD18-mediated ubiquitylation of PCNA was RPA-insensitive. Our findings suggest that RPA plays a regulatory role in DNA damage responses via repression of RFC2 ubiquitylation in human cells.
In eukaryotic cells, DNA replication is carried out by coordinated actions of many proteins, including DNA polymerase delta (pol delta), replication factor C (RFC), proliferating cell nuclear antigen (PCNA) and replication protein A. Here we describe dynamic properties of these proteins in the elongation step on a single-stranded M13 template, providing evidence that pol delta has a distributive nature over the 7 kb of the M13 template, repeating a frequent dissociation-association cycle at growing 3'-hydroxyl ends. Some PCNA could remain at the primer terminus during this cycle, while the remainder slides out of the primer terminus or is unloaded once pol delta has dissociated. RFC remains around the primer terminus through the elongation phase, and could probably hold PCNA from which pol delta has detached, or reload PCNA from solution to restart DNA synthesis. Furthermore, we suggest that a subunit of pol delta, POLD3, plays a crucial role in the efficient recycling of PCNA during dissociation-association cycles of pol delta. Based on these observations, we propose a model for dynamic processes in elongation complexes.
