Control of Genome Integrity by RFC Complexes; Conductors of PCNA Loading onto and Unloading from Chromatin during DNA Replication

Abstract

During cell division, genome integrity is maintained by faithful DNA replication during S phase, followed by accurate segregation in mitosis. Many DNA metabolic events linked with DNA replication are also regulated throughout the cell cycle. In eukaryotes, the DNA sliding clamp, proliferating cell nuclear antigen (PCNA), acts on chromatin as a processivity factor for DNA polymerases. Since its discovery, many other PCNA binding partners have been identified that function during DNA replication, repair, recombination, chromatin remodeling, cohesion, and proteolysis in cell-cycle progression. PCNA not only recruits the proteins involved in such events, but it also actively controls their function as chromatin assembles. Therefore, control of PCNA-loading onto chromatin is fundamental for various replication-coupled reactions. PCNA is loaded onto chromatin by PCNA-loading replication factor C (RFC) complexes. Both RFC1-RFC and Ctf18-RFC fundamentally function as PCNA loaders. On the other hand, after DNA synthesis, PCNA must be removed from chromatin by Elg1-RFC. Functional defects in RFC complexes lead to chromosomal abnormalities. In this review, we summarize the structural and functional relationships among RFC complexes, and describe how the regulation of PCNA loading/unloading by RFC complexes contributes to maintaining genome integrity.

Keywords: Ctf18; DNA replication; Elg1; PCNA; PCNA loader; PCNA unloader; RFC complex; RFC1; chromatin; genome integrity.

Figure 1

Summary of the functions of the three RFC complexes transacting on PCNA [17]. See text for details. The “?” marks in the table mean that the main effect of Ctf18-RFC (loading and unloading) on PCNA in vivo is not well understood.

Figure 2

PCNA loading on and unloading from chromatin by RFC complexes during DNA synthesis. ① PCNA loader RFC1-RFC or Ctf18-RFC bind to PCNA and recognize the 3′ DNA template, and ATP binding triggers a conformational change of the RFC complex that allows for a tight interaction with PCNA and ring opening. ② ATP hydrolysis by the RFC loader complex is coupled with ring closure and the release of PCNA, finally encircling the DNA duplex. ③ DNA polymerases bind to chromatin-loaded PCNA, and DNA synthesis begins. ④ After the DNA synthesis is complete and DNA polymerase is released, PCNA recruits various enzymes for additional functions such as chromatin remodeling. PCNA slides along the double-stranded DNA to its functional sites. In ③ and ④, PCNA might be modified by mono-ubiquitin, poly-ubiquitin, SUMO (small ubiquitin-like modifier), or acetyl depending on the circumstances as illustrated in a dotted-line square (note that modification on single subunit of PCNA trimer is shown). ⑤ After the role of PCNA is completed, Elg1-RFC unloads PCNA from the double-stranded DNA in an ATP-dependent manner as a reverse reaction of PCNA loading. ⑥ During PCNA unloading, its modification might be removed so that it can be recycled. In this figure, nucleosomes and chromatin structures are omitted.

Figure 3

RFC subunit structures. The center of these subunits includes RFC boxes containing a P-loop, which is a general Walker-type ATPase motif. The C-terminal regions following the grey boxes representing the four small subunits (RFC2–5) are required for RFC complex formation. As for the largest RFC subunits, the domains required for complex formation are not well defined. The N-terminal of RFC1 contains a BRCT motif and the C-terminal of Ctf18 contains an interaction motif with Dcc1 and Ctf8. The N-terminal of Elg1 includes a SUMO (in Sc, called SIM) or UAF1 (in Hs) binding motif, which is potentially involved in PCNA binding. The UAF1 binding motif in Hs Elg1 likely has a role as a SIM, thus referred as “SIM?”. Hs: Homo sapiens, Sc: Saccharomyces cerevisiae.

Figure 4

PCNA loading or unloading function of human RFC complexes. (A) Depletion by RNA interference (RNAi) or overexpression (OE) of the largest subunits of RFCs in human HEK293 cells. Whole cell extract (WCE) and chromatin-containing fractions (Chr) were prepared after centrifugation. The results demonstrated that RFC1-RFC and Elg1-RFC have a primary role in PCNA loading and unloading, respectively, in vivo. Depletion or overexpression of Ctf18 does not change the level of PCNA on chromatin in human cells; (B) PCNA unloading assay. Left panel: partially purified Elg1-RFC complex. HEK293T cells were co-transfected with FLAG-tagged Elg1 and RFC2-5, and complexes were purified with anti-FLAG antibody. Right panel: PCNA unloading assay. The purified Elg1-RFC was incubated with permeabilized cell nuclei containing PCNA-loaded chromatin in the presence or absence of ATP. The purified Elg1-RFC complexes unload PCNA from chromatin in an ATP-dependent manner.