A charged residue at the subunit interface of PCNA promotes trimer formation by destabilizing alternate subunit interactions

Freudenthal, Bret; Gakhar, Lokesh; Ramaswamy, S.; Washington, M. Todd

doi:10.1107/s0907444909011329

Cited by 14 publications

(25 citation statements)

References 19 publications

(34 reference statements)

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“…As PCNA-C81R and an unrelated trimer interface mutant have trimerization defects (Freudenthal et al, 2009; Lau et al, 2002), we fractionated the mutant PCNA proteins on a size-exclusion column at a concentration of 2.88 μM. Wild-type PCNA had a retention time of 35 minutes, corresponding to a Stokes radius of 49 Å (Figure 2A).…”

Section: Resultsmentioning

confidence: 99%

PCNA and Msh2-Msh6 Activate an Mlh1-Pms1 Endonuclease Pathway Required for Exo1-Independent Mismatch Repair

et al. 2014

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Summary Genetic evidence has implicated multiple pathways in eukaryotic DNA mismatch repair (MMR) downstream of mispair recognition and Mlh1-Pms1 recruitment, including Exonuclease 1 (Exo1) dependent and independent pathways. We identified 14 mutations in POL30, which encodes PCNA in Saccharomyces cerevisiae, specific to Exo1-independent MMR. The mutations identified affected amino acids at three distinct sites on the PCNA structure. Multiple mutant PCNA proteins had defects either in trimerization and Msh2-Msh6 binding or in activation of the Mlh1-Pms1 endonuclease that initiates excision during MMR. The latter class of mutations led to hyper-accumulation of repair intermediate Mlh1-Pms1 foci and were enhanced by an msh6 mutation that disrupted the Msh2-Msh6 interaction with PCNA. These results reveal a central role for PCNA in the Exo1-independent MMR pathway and suggest that Msh2-Msh6 localizes PCNA to repair sites after mispair recognition to activate the Mlh1-Pms1 endonuclease for initiating Exo1-dependent repair or for driving progressive excision in Exo1-independent repair.

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Section: Resultsmentioning

confidence: 99%

PCNA and Msh2-Msh6 Activate an Mlh1-Pms1 Endonuclease Pathway Required for Exo1-Independent Mismatch Repair

et al. 2014

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“…It was, therefore, suggested that the G178S mutation prevented ubiquitylation of Lys-164 in PCNA (41), although this has not been directly tested. In another study, a crystal structure was reported for a PCNA variant carrying one of the two amino acid substitutions present in pol30-113, E113G (42). The structural change in the intermolecular interface region brought about by the E113G substitution was found to be similar to the one in the G178S variant (43).…”

Section: Discussionmentioning

confidence: 96%

“…In addition, the absence of Pol stimulation by PCNA-E113G could reflect the fact that this PCNA variant is expected to have a severe defect in the trimer stability under the assay conditions used in that study (a low PCNA concentration and no macromolecular crowding agent; Refs. 42,43). Future studies of PCNA-113 structure could help characterize the changes in the protein that alter its interactions with TLS polymerases.…”

Section: Discussionmentioning

confidence: 99%

The Non-canonical Protein Binding Site at the Monomer-Monomer Interface of Yeast Proliferating Cell Nuclear Antigen (PCNA) Regulates the Rev1-PCNA Interaction and Polζ/Rev1-dependent Translesion DNA Synthesis

Sharma¹,

Kochenova

Shcherbakova

2011

Journal of Biological Chemistry

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Rev1 and DNA polymerase (Pol) are involved in the tolerance of DNA damage by translesion synthesis (TLS). The proliferating cell nuclear antigen (PCNA), the auxiliary factor of nuclear DNA polymerases, plays an important role in regulating the access of TLS polymerases to the primer terminus. Both Rev1 and Pol lack the conserved hydrophobic motif that is used by many proteins for the interaction with PCNA at its interdomain connector loop. We have previously reported that the interaction of yeast Pol with PCNA occurs at an unusual site near the monomer-monomer interface of the trimeric PCNA. Using GST pull-down assays, PCNA-coupled affinity beads pulldown and gel filtration chromatography, we show that the same region is required for the physical interaction of PCNA with the polymerase-associated domain (PAD) of Rev1. The interaction is disrupted by the pol30-113 mutation that results in a double amino acid substitution at the monomer-monomer interface of PCNA. Genetic analysis of the epistatic relationship of the pol30-113 mutation with an array of DNA repair and damage tolerance mutations indicated that PCNA-113 is specifically defective in the Rev1/Pol-dependent TLS pathway. Taken together, the data suggest that Pol and Rev1 are unique among PCNA-interacting proteins in using the novel binding site near the intermolecular interface of PCNA. The new mode of Rev1-PCNA binding described here suggests a mechanism by which Rev1 adopts a catalytically inactive configuration at the replication fork.Cellular DNA is continuously attacked by endogenous and exogenous agents that damage the bases and the DNA backbone. Damage that is not repaired prior to the S phase of cell cycle can block the replication machinery, because most lesions cannot be accommodated in the highly selective active site of replicative DNA polymerases (1, 2). Replication stalling activates several damage tolerance mechanisms that help bypass the damage in either an accurate or mutagenic manner. Translesion DNA synthesis (TLS) 3 is an important damage tolerance pathway, in which specialized DNA polymerases are recruited to synthesize DNA through template lesions (3). Structural studies showed that TLS polymerases have a more open active site that allows them to accommodate a variety of DNA lesions and catalyze polymerization on damaged templates (4). The accuracy of TLS can vary depending on the particular lesion and the DNA polymerases involved. It is, however, an inherently mutagenic process, because the damage can alter the coding properties of the bases, and because TLS polymerases generally have low fidelity (2).In human cells, TLS polymerases include Y family enzymes Pol, Pol, Pol, and REV1, and the B family enzyme Pol. Although the Y family polymerases share little amino acid sequence similarity with the classical DNA polymerases, they have a similar overall "right-hand" architecture with the "palm," "thumb," and "fingers" domains. The thumb and fingers domains of the Y family enzymes, however, are short and do not make as many contacts wi...

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“…Both of these substitutions (G178S and E113G) are of residues in the β strands that constitute the subunit interface, and the X-ray crystal structures of both mutant proteins reveal perturbations that reduce the number of hydrogen bonds between these strands [90, 91]. The mutant PCNA trimers are less stable than wild-type PCNA, and they are unable to stimulate the catalytic activity of non-classical polymerases [87].…”

Section: The Role Of Pcna In Translesion Synthesismentioning

confidence: 99%

The Many Roles of PCNA in Eukaryotic DNA Replication

2016

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Proliferating cell nuclear antigen (PCNA) plays critical roles in many aspects of DNA replication and replication-associated processes, including translesion synthesis, error-free damage bypass, break-induced replication, mismatch repair, and chromatin assembly. Since its discovery, our view of PCNA has evolved from a replication accessory factor to the hub protein in a large protein-protein interaction network that organizes and orchestrates many of the key events at the replication fork. We begin this review article with an overview of the structure and function of PCNA. We discuss the ways its many interacting partners bind and how these interactions are regulated by post-translational modifications such as ubiquitylation and sumoylation. We then explore the many roles of PCNA in normal DNA replication and in replication-coupled DNA damage tolerance and repair processes. We conclude by considering how PCNA can interact physically with so many binding partners to carry out its numerous roles. We propose that there is a large, dynamic network of linked PCNA molecules at and around the replication fork. This network would serve to increase the local concentration of all the proteins necessary for DNA replication and replication-associated processes and to regulate their various activities.

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A charged residue at the subunit interface of PCNA promotes trimer formation by destabilizing alternate subunit interactions

Cited by 14 publications

References 19 publications

PCNA and Msh2-Msh6 Activate an Mlh1-Pms1 Endonuclease Pathway Required for Exo1-Independent Mismatch Repair

PCNA and Msh2-Msh6 Activate an Mlh1-Pms1 Endonuclease Pathway Required for Exo1-Independent Mismatch Repair

The Non-canonical Protein Binding Site at the Monomer-Monomer Interface of Yeast Proliferating Cell Nuclear Antigen (PCNA) Regulates the Rev1-PCNA Interaction and Polζ/Rev1-dependent Translesion DNA Synthesis

The Many Roles of PCNA in Eukaryotic DNA Replication

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