Deletion Analysis of the Large Subunit p140 in Human Replication Factor C Reveals Regions Required for Complex Formation and Replication Activities

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The level of monoubiquitinated proliferating cell nuclear antigen (PCNA) is closely linked with DNA damage bypass to protect cells from a high level of mutagenesis. However, it remains unclear how the level of monoubiquitinated PCNA is regulated. Here, we demonstrate that human ELG1 protein, which comprises an alternative replication factor C (RFC) complex and plays an important role in preserving genomic stability, as an interacting partner for the USP1 (ubiquitin-specific protease 1)-UAF1 (USP1-associated factor 1) complex, a deubiquitinating enzyme complex for PCNA and FANCD2. ELG1 protein interacts with PCNAs that are localized at stalled replication forks. ELG1 knockdown specifically resulted in an increase in the level of PCNA monoubiquitination without affecting the level of FANCD2 ubiquitination. It is a novel function of ELG1 distinct from its role as an alternative RFC complex because knockdowns of any other RFC subunits or other alternative RFCs did not affect PCNA monoubiquitination. Lastly, we identified a highly conserved N-terminal domain in ELG1 that was responsible for the USP1-UAF1 interaction as well as the activity to down-regulate PCNA monoubiquitination. Taken together, ELG1 specifically directs USP1-UAF1 complex for PCNA deubiquitination. Proliferating cell nuclear antigen (PCNA)4 functions in DNA replication, repair, and recombination as a sliding clamp for various DNA replication polymerases and a scaffold for many DNA repair and recombination enzymes (1). Various posttranslational modifications of PCNA, including ubiquitination, sumoylation, or phosphorylation, determine its specific functions (2, 3). PCNA ubiquitination directs different branches of the postreplication repair (PRR) pathway (4). When a DNA replication fork stalls at damaged DNA lesions, the ubiquitin conjugation enzyme, RAD6, and the ubiquitin ligase, RAD18, monoubiquitinate lysine 164 of PCNA (5). Monoubiquitinated PCNA recruits translesion synthesis (TLS) polymerases, which can insert a base opposite the damaged lesion to bypass the damaged DNA. Alternatively, PCNA can be polyubiquitinated to promote DNA damage bypass by a homologous recombination mechanism (6 -8).The process and consequence of PCNA deubiquitination after DNA damage bypass are not clearly understood. Recently, USP1 (ubiquitin-specific protease 1) was identified as a deubiquitinating enzyme for PCNA after DNA damage bypass (9, 10). USP1 reduces the accumulation of monoubiquitinated PCNA during normal cell division, which prevents mutagenesis by error-prone TLS polymerases. In response to damage that stalls DNA replication, USP1 is degraded, and consequently monoubiquitinated PCNA accumulates (9). However, the observation that levels of monoubiquitinated PCNA remain high, even in the presence of persistent levels of USP1 (11,12) suggests that there might be additional mechanisms to regulate USP1 activity. The identification of UAF1 (USP1-associated factor 1) indicates that interacting proteins may regulate the activity of USP1 in vivo (13).The loading a...

Section: Discussionmentioning

confidence: 80%

Human ELG1 Regulates the Level of Ubiquitinated Proliferating Cell Nuclear Antigen (PCNA) through Its Interactions with PCNA and USP1

Lee

Yang

Cohn

et al. 2010

116

157

“…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.…”

Section: Resultsmentioning

confidence: 99%

“…The studies of this report do not rule out contact between PCNA and RFC2 or 5, as only strongly interacting complexes are observed by the non-equilibrium gel filtration technique used here. Indeed it has been suggested that all five RFC subunits contact PCNA in the human system (32).…”

Section: Resultsmentioning

confidence: 99%

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

Yao

Coryell

Zhang

et al. 2003

Self Cite

Replication factor C (RFC) is a heteropentameric AAA؉ protein clamp loader of the proliferating cell nuclear antigen (PCNA) processivity factor. The prokaryotic homologue, ␥ complex, is also a heteropentamer, and structural studies show the subunits are arranged in a circle. In this report, Saccharomyces cerevisiae RFC protomers are examined for their interaction with each other and PCNA. The data lead to a model of subunit order around the circle. A characteristic of AAA؉ oligomers is the use of bipartite ATP sites in which one subunit supplies a catalytic arginine residue for hydrolysis of ATP bound to the neighboring subunit. We find that the RFC(3/4) complex is a DNA-dependent ATPase, and we use this activity to determine that RFC3 supplies a catalytic arginine to the ATP site of RFC4. This information, combined with the subunit arrangement, defines the composition of the remaining ATP sites. Furthermore, the RFC(2/3) and RFC(3/4) subassemblies bind stably to PCNA, yet neither RFC2 nor RFC4 bind tightly to PCNA, indicating that RFC3 forms a strong contact point to PCNA. The RFC1 subunit also binds PCNA tightly, and we identify two hydrophobic residues in RFC1 that are important for this interaction. Therefore, at least two subunits in RFC make strong contacts with PCNA, unlike the Escherichia coli ␥ complex in which only one subunit makes strong contact with the ␤ clamp. Multiple strong contact points to PCNA may reflect the extra demands of loading the PCNA trimeric ring onto DNA compared with the dimeric ␤ ring.Replicases of cellular chromosomes utilize a circular sliding clamp protein that encircles DNA and tethers the polymerase to the template for high processivity in DNA synthesis (1). An example of this protein class is the Escherichia coli ␤ subunit, which confers high processivity onto the chromosomal replicase, DNA polymerase III holoenzyme (2, 3). The eukaryotic equivalent is the PCNA 1 ring, which has essentially the same shape and chain fold as ␤ despite lack of sequence similarity between the two (4, 5). These ring-shaped proteins require an ATP-fueled multiprotein clamp loader for assembly onto primed DNA.The eukaryotic clamp loader is the heteropentameric replication factor C (RFC). The five subunits of RFC are each different proteins, but they are homologous to one another (6, 7) and are members of the AAAϩ family of ATPases (8). The recent crystal structure of E. coli ␥ complex, the prokaryotic counterpart of RFC, has facilitated detailed hypothesis regarding RFC structure and mechanism (9, 10). The ␥ complex (␥ 3 ␦␦Ј ) consists of a minimal core of five proteins (␥ 3 ␦␦Ј), which contain the clamp loading activity (11). The remaining two subunits, and , are involved in recruiting an RNA primed DNA site from the primase, and they bind singlestranded DNA-binding protein (SSB) to assist polymerase elongation but are not essential to the clamp loading activity of ␥ complex (12)(13)(14). Biochemical studies of ␥ complex (15-17), combined with crystal structures of ␥ 3 ␦␦Ј (10) and ␦-␤ 1 complex ...

“…The other four RFC subunits, referred to as the small subunits, are in the 36 -40-kDa range: yeast Rfc2(D) (human p37), yeast Rfc3(C) (human p36), yeast Rfc4(B) (human p40), and yeast Rfc5(E) (human p38). The N-terminal region of Rfc1, up to the region of homology to the other RFC subunits, can be deleted in both yeast and human RFC without decreasing RFC clamp loader function with PCNA (10,11). Alternative clamp loaders exist in which Rfc1 is replaced by another protein.…”

mentioning

confidence: 99%

Mechanism of Proliferating Cell Nuclear Antigen Clamp Opening by Replication Factor C

Yao

Johnson

Bowman

et al. 2006

Self Cite

The eukaryotic replication factor C (RFC) clamp loader is an AAA؉ spiral-shaped heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) clamp processivity factor on DNA. In this study, we examined the roles of individual RFC subunits in opening the PCNA clamp. Interestingly, Rfc1, which occupies the position analogous to the ␦ clamp-opening subunit in the Escherichia coli clamp loader, is not required to open PCNA. The Rfc5 subunit is required to open PCNA. Consistent with this result, Rfc2⅐3⅐4⅐5 and Rfc2⅐5 subassemblies are capable of opening and unloading PCNA from circular DNA. Rfc5 is positioned opposite the PCNA interface from Rfc1, and therefore, its action with Rfc2 in opening PCNA indicates that PCNA is opened from the opposite side of the interface that the E. coli ␦ wrench acts upon. This marks a significant departure in the mechanism of eukaryotic and prokaryotic clamp loaders. Interestingly, the Rad⅐RFC DNA damage checkpoint clamp loader unloads PCNA clamps from DNA. We propose that Rad⅐RFC may clear PCNA from DNA to facilitate shutdown of replication in the face of DNA damage.The proliferating cell nuclear antigen (PCNA) 3 DNA sliding clamp is a ring-shaped homotrimer that is opened and closed around DNA by the replication factor C (RFC) clamp loader (1-4). The PCNA clamp then binds numerous different proteins, including the cellular replicases DNA polymerase ␦ and DNA polymerase ⑀ (5). The RFC clamp loader was initially identified as a factor required for SV40 DNA replication in vitro (2). Subsequent characterization of RFC showed it to consist of five different subunits in both yeast and human cells (6). The five subunits are homologous to one another and are members of the AAAϩ family of ATPases (7). The structures of yeast RFC and the Escherichia coli clamp loader ␥ complex reveal that the subunits are arranged in a similar spiral fashion (8, 9). To avoid confusion over the different nomenclatures used for the different systems, the positions of the various clamp loader subunits are designated A-E as listed in Table 1. The position of each subunit in the complex is indicated in parentheses with a letter when comparisons are made between systems.The yeast Rfc1(A) subunit (human p140(A)) is sometimes referred to as the large subunit, as it contains both N-and C-terminal extensions past its region of homology with the four small subunits. The other four RFC subunits, referred to as the small subunits, are in the 36 -40-kDa range: yeast Rfc2(D) (human p37), yeast Rfc3(C) (human p36), yeast Rfc4(B) (human p40), and yeast Rfc5(E) (human p38). The N-terminal region of Rfc1, up to the region of homology to the other RFC subunits, can be deleted in both yeast and human RFC without decreasing RFC clamp loader function with PCNA (10, 11). Alternative clamp loaders exist in which Rfc1 is replaced by another protein. An example of one such alternative clamp loader is the Rad⅐RFC DNA damage checkpoint clamp loader, in which Rad24(A) (Rad17 in humans) replaces Rfc1(A) and...